Bacteria separation system and methods

ABSTRACT

Methods and apparatus for detecting, quantifying, enriching, and/or separating bacterial species in fluid sample are provided. The fluid sample is provided as input to a microfluidic passage of a microfluidic device, wherein the microfluidic device comprises at least one electrode disposed adjacent to the microfluidic passage. The at least one electrode is activated to capture bacteria in the sample using dielectrophoresis, wherein the capture efficiency of bacteria is at least 99%.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationSerial No. PCT/US2020/060412, filed Nov. 13, 2020, entitled “METHODS ANDAPPARATUS FOR DETECTION OF BACTERIA IN A SAMPLE USINGDIELECTROPHORESIS”, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 62/934,856 filed Nov. 13, 2019 andentitled BACTERIAL AND VIRAL TESTING IN 30 MINUTES WITH AN AI ENABLEDMICROCHIP,” the entire contents of which is incorporated by referenceherein

BACKGROUND

Detection and identification of bacterial and viral pathogens present incell containing solutions (e.g., blood, urine, CSF), protein containingsolutions (e.g., for quality control in pharmaceuticals duringmanufacturing), analyte extraction from microbiome samples, water,sterile fluids and other fluids is possible by employing isolation oncultural media and metabolic fingerprinting methods. Isozome analysis,direct colony thin layer chromatography and gel electrophoresistechniques have been successfully applied for the detection of somebacterial pathogens. Immunoassay and nucleic acid-based assays are nowwidely accepted techniques, providing more sensitive and specificdetection and quantification of bacterial pathogens.

Dielectrophoresis (DEP) relates to a force of an electric field gradienton objects having dielectric moments. DEP has shown promise for particleseparation, but has not yet been applied in clinical settings. DEP usesa natural or induced dipole to cause a net force on a particle in aregion having an electric field gradient. The force depends on theClausius-Mossotti factor associated with particle.

SUMMARY

Some embodiments relate to a method of high-efficiency capture ofbacteria in sample. The method comprises providing the sample as inputto a microfluidic passage of a microfluidic device at a predeterminedflow rate, wherein the microfluidic device comprises at least oneelectrode disposed adjacent to the microfluidic passage and activatingthe at least one electrode to capture bacteria in the sample by the atleast one electrode using dielectrophoresis as the sample flows throughthe microfluidic passage at the predetermined flow rate, wherein thecapture efficiency of bacteria is at least 99% when the predeterminedflow rate is between 10-960 ul/min.

In at least one aspect, the capture efficiency of bacteria is at least99% when the predetermined flow rate is between 480-960 ul/min. In atleast one aspect, the capture efficiency of bacteria is at least 99%when the predetermined flow rate is between 720-960 ul/min. In at leastone aspect, the capture efficiency of bacteria is at least 99% when thepredetermined flow rate is between 840-960 ul/min. In at least oneaspect, the capture efficiency of bacteria is at least 99.6%. In atleast one aspect, the capture efficiency of bacteria is at least 99.9%when the predetermined flow rate is between 240-480 ul/min. In at leastone aspect, the capture efficiency of bacteria is at least 99.99% whenthe predetermined flow rate is 240 ul/min.

In at least one aspect, the method further comprises quantifying, usingan optical system, an amount of bacteria captured by the at least oneelectrode during activation of the at least one electrode. In at leastone aspect, quantifying the amount of bacteria comprises capturing oneor more images using the optical system, and processing, using at leastone computing device, the one or more images to quantify the amount ofbacteria. In at least one aspect, quantifying the amount of bacteriacomprises counting a number of spots in one or more images captured bythe optical system.

In at least one aspect, the method further comprises labeling thebacteria captured on the at least one electrode with a fluorescent dye,exciting the fluorescent dye with at least one light source of theoptical system to produce a fluorescent signal, and capturing thefluorescent signal using the optical system, and quantifying the amountof bacteria based, at least in part, on the captured fluorescent signal.

In at least one aspect, the method further comprises collecting effluentfluid at an output of the microfluidic channel, determining an amount ofbacteria in the collected effluent fluid, and calculating the captureefficiency based, at least in part, on the determined amount of bacteriain the effluent fluid. In at least one aspect, calculating the captureefficiency based, at least in part, on the determined amount of bacteriain the effluent fluid comprises comparing a concentration of bacteria inthe sample provided as input to the microfluidic channel and aconcentration of bacteria in the collected effluent fluid.

In at least one aspect, activating the at least one electrode comprisesapplying an alternating current (AC) voltage to the at least oneelectrode, wherein the AC voltage has a frequency between 900 Hz and 2MHz. In at least one aspect, the AC voltage has a frequency of 1 MHz.

In at least one aspect, the at least one electrode comprises a pluralityof concentric rings or arcs.

In at least one aspect, the microfluidic passage comprises amicrofluidic channel formed in a microfluidic chip.

In at least one aspect, the method further comprises altering acharacteristic of an AC voltage provided to activate the at least oneelectrode, wherein altering the characteristic of the AC voltage causesthe capture bacteria to be released from the at least one electrode. Inat least one aspect, the characteristic is a frequency of the ACvoltage. In at least one aspect, altering the frequency of the ACvoltage comprises providing a higher frequency AC voltage to the atleast one electrode to apply negative dielectrophoresis to the bacteria.In at least one aspect, the characteristic is an amplitude of the ACvoltage.

In at least one aspect, the method further comprises flushing a buffersolution through the microfluidic passage to mechanically release thebacteria from the at least one electrode.

Some embodiments relate to a bacterial detection system. The bacterialdetection system comprises a microfluidic device including amicrofluidic passage having an inlet and an outlet and at least oneelectrode disposed adjacent to the microfluidic passage, wherein the atleast one electrode when activated, is configured to capture, usingdielectrophoresis, bacteria in a sample flowing through the microfluidicpassage at a predetermined flow rate, and wherein a capture efficiencyof bacteria by the at least one electrode is at least 99% when thepredetermined flow rate is between 10-960 ul/min.

In at least one aspect, the bacterial detection system further comprisesa first pump coupled to the microfluidic passage, wherein first pump isconfigured to pump the sample through the microfluidic passage at thepredetermined flow rate. In at least one aspect, the first pump iscoupled to the outlet of the microfluidic passage. In at least oneaspect, the first pump is coupled to the inlet of the microfluidicpassage.

In at least one aspect, the bacterial detection system further comprisesa second pump coupled to the output of the microfluidic passage, whereinthe second pump is configured to pump the sample out of the microfluidicpassage.

In at least one aspect, the bacterial detection system further comprisesan optical system configured to capture one or more images of the atleast one electrode during capture of the bacteria.

In at least one aspect, the bacterial detection system further comprisesat least one computing device configured to process the one or moreimages to quantify an amount of bacteria captured by the at least oneelectrode.

In at least one aspect, the at least one electrode comprises an array ofelectrodes arranged in at least one dimension along the microfluidicpassage. In at least one aspect, the array of electrodes is arranged inat least two dimensions along the microfluidic passage.

In at least one aspect, the bacterial detection system further comprisesa signal generator configured to activate the at least one electrode byapplying an alternating current (AC) voltage thereto to generate anelectric field. In at least one aspect, the signal generator isconfigured to apply a same AC voltage to each of the electrodes in thearray of electrodes. In at least one aspect, the signal generator isconfigured to apply a first AC voltage to a first electrode in the arrayof electrodes and a second AC voltage to a second electrode in the arrayof electrodes, the first AC voltage and the second AC voltage beingdifferent. In at least one aspect, the first AC voltage and the secondAC voltage have a different amplitude and/or frequency.

In at least one aspect, the microfluidic passage comprises amicrofluidic channel.

In at least one aspect, the microfluidic device comprises a microfluidicchip having a plurality of microfluidic passages configured to process aplurality of samples in parallel.

Some embodiments relate to a method of quantifying bacteria in sample.The method comprises providing, in a first run, a first portion of thesample as input to a microfluidic channel of a microfluidic chip,wherein the microfluidic chip comprises at least one electrode disposedadjacent to the microfluidic channel, activating the at least oneelectrode to capture bacteria in the first portion of the sample by theat least one electrode using dielectrophoresis, quantifying a firstamount of bacteria captured by the at least one electrode duringactivation of the at least one electrode during the first run,providing, in a second run, a second portion of the sample as input tothe microfluidic channel of the microfluidic chip, activating the atleast one electrode to capture bacteria in the second portion of thesample by the at least one electrode using dielectrophoresis, andquantifying a second amount of bacteria captured by the at least oneelectrode during activation of the at least one electrode during thesecond run, wherein the first amount and second amount are within +/−0.5log.

In at least one aspect, the method further comprises performing at leastten runs including the first run and the second run and quantifying anamount of bacteria in each of the at least ten runs, wherein an amountof variability in the quantified amount of bacteria across the at leastten runs is less than +/−0.5 log.

Some embodiments relate to a bacterial capture system. The bacterialcapture system comprising a microfluidic chip including a microfluidicpassage and at least one electrode disposed adjacent to the microfluidicpassage, wherein the at least one electrode when activated, isconfigured to capture, using dielectrophoresis, bacteria in a sampleflowing through the microfluidic passage, and wherein a variability ofan amount of bacteria captured across multiple runs of the sampleflowing through the microfluidic passage is less than +/−0.5 log.

Some embodiments relate to a method for enriching a bacterial species ina sample containing a first target bacterial species and othercomponents. The method comprises providing the sample as input to amicrofluidic passage included as a portion of a microfluidic device,wherein the microfluidic passage has at least one electrode disposedadjacent thereto, selecting at least one characteristic of an AC voltageapplied to the at least one electrode, wherein the selection of the atleast one characteristic is based, at least in part, on the first targetbacterial species, applying the AC voltage having the selected at leastone characteristic to the at least one electrode to generate an electricfield that produces a positive dielectrophoresis force to capture on asurface of the at least one electrode, the first target bacterialspecies as the sample flows through the microfluidic channel, releasingthe captured first target bacterial species from the at least oneelectrode, and collecting effluent fluid including the captured firsttarget bacterial species, wherein a relative abundance of the firsttarget bacterial species in the effluent fluid is increased compared tothe relative abundance of the first target bacterial species in thesample.

In at least one aspect, the other components include a second targetbacterial species, and the selection of the at least one characteristicis further based, at least in part, on the second target bacterialspecies such that both the first and the second target bacterial speciesare captured on the surface of the at least one electrode as the sampleflows through the microfluidic channel when the AC voltage is applied tothe at least one electrode.

In at least one aspect, releasing the captured first target bacterialspecies from the at least one electrode comprises releasing only thefirst target bacterial species captured on the surface of the at leastone electrode.

In at least one aspect, releasing only the first target bacterialspecies captured on the surface of the at least one electrode comprisesadjusting a frequency of the AC voltage applied to the at least oneelectrode such that a negative dielectrophoresis force is applied to thefirst target bacterial species to cause the first target bacterialspecies to be released from the surface of the at least one electrode.

In at least one aspect, adjusting a frequency of the AC voltagecomprises increasing the frequency of the AC voltage.

In at least one aspect, collecting effluent fluid including the capturedfirst target bacterial species comprises collecting effluent fluidincluding only the captured first target bacterial species.

In at least one aspect, the method further comprises releasing allremaining bacterial species captured on the surface of the at least oneelectrode.

In at least one aspect, releasing all remaining bacterial speciescaptured on the at least one electrode comprises turning deactivatingthe at least one electrode.

In at least one aspect, releasing all remaining bacterial speciescaptured on the at least one electrode comprises mechanically releasingall remaining bacterial species.

In at least one aspect, mechanically releasing all remaining bacterialspecies comprises flushing the microfluidic channel with a fluid.

In at least one aspect, the other components include a second targetbacterial species, and the selection of the at least one characteristicof the AC voltage is further based, at least in part, on the secondtarget bacterial species such that first target bacterial species iscaptured on the surface of the at least one electrode as the sampleflows through the microfluidic passage and the second target bacterialspecies is not captured on the surface of the at least one electrode.

In at least one aspect, the at least one characteristic is an amplitudeand/or a frequency of the AC voltage.

In at least one aspect, the at least one characteristic comprises theamplitude and the frequency of the AC voltage.

In at least one aspect, the method further comprises pumping the samplethrough the microfluidic passage.

In at least one aspect, the one or more bacterial species include grampositive bacterial species and gram negative bacterial species, andwherein selecting at least one characteristic of an AC voltage appliedto the at least one electrode comprises selecting the at least onecharacteristic of the AC voltage such than only one of the gram positivebacterial species and the gram negative bacterial species is captured bythe at least one electrode.

In at least one aspect, a relative abundance of the first targetbacterial species in the effluent fluid is increased at least 20 timescompared to the relative abundance of the first target bacterial speciesin the sample.

In at least one aspect, the sample is a fecal sample or a microbiomesample.

In at least one aspect, the relative abundance of the first targetbacterial species in the sample is below a detection limit of DNAsequencing.

In at least one aspect, the first target bacterial species compriseslive bacteria and the other components include dead bacteria.

In at least one aspect, quantifying an amount of first target bacterialspecies captured on the surface of the at least one electrode.

Some embodiments relate to a bacterial enrichment system. The bacterialenrichment system comprising a microfluidic chip including amicrofluidic passage and at least one electrode disposed adjacent to themicrofluidic passage, a pump coupled to the microfluidic chip andconfigured to pump a sample from an inlet of the microfluidic passage toan outlet of the microfluidic passage, a signal generator electricallyconnected to the at least one electrode and configured to generate an ACvoltage to drive the at least one electrode to produce an electric fieldwithin the microfluidic passage, and a controller configured to controlthe signal generator to generate the AC voltage having frequency andamplitude characteristics such that when produced, the electric fieldcaptures on the surface of the at least one electrode, a targetbacterial species in the sample while not capturing one or more othercomponents in a sample as the sample is pumped through the microfluidicchannel, and control the signal generator to alter generation of the ACvoltage to release the captured target bacterial species in the sample.

In at least one aspect, the pump is coupled to the inlet of the at leastone microfluidic channel.

In at least one aspect, the pump is coupled to an outlet of the at leastone microfluidic channel.

In at least one aspect, the pump is coupled to the microfluidic chipoutside of flow path of the sample.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various non-limiting embodiments of the technology will be describedwith reference to the following figures. It should be appreciated thatthe figures are not necessarily drawn to scale.

FIG. 1 illustrates a plate counting technique for detecting bacteria ina sample;

FIG. 2 illustrates a system for detecting the presence of bacteria in asample, according to some embodiments;

FIG. 3 illustrates a microfluidic system for detecting the presence ofbacteria in a sample, according to some embodiments;

FIG. 4 illustrates a flow-based system for detecting the presence ofbacteria in a fluid sample, according to some embodiments;

FIG. 5 illustrates a static system for detecting the presence ofbacteria in a fluid sample, according to some embodiments;

FIG. 6A illustrates a static system in operation for capturing bacteriain a sample, according to some embodiments;

FIG. 6B illustrates a flow-based system in operation for capturingbacteria in a sample, according to some embodiments;

FIG. 7A illustrates a schematic of an electrode configuration that maybe used to capture bacteria in accordance with some embodiments;

FIGS. 7B and 7C illustrate zoomed-in versions of the electrodeconfiguration of FIG. 7A;

FIGS. 7D and 7E show operation of the electrode configuration of FIG. 7Ain the absence and presence of an electric field, respectively, inaccordance with some embodiments;

FIG. 7F shows an image of an entire electrode configuration havingbacteria captured thereon in accordance with some embodiments;

FIG. 8 is a flow chart of a process for detecting and quantifyingbacteria in a static fluid sample in accordance with some embodiments;

FIG. 9 shows results of an experiment for quantifying bacteria in asample using the process of FIG. 8;

FIGS. 10A-10C show results of experiments for quantifying bacteria in asample using multiple microfluidic devices using the process of FIG. 8;

FIG. 11 is a flow chart of a process for detecting and quantifyingbacteria in a flowing fluid sample in accordance with some embodiments;

FIG. 12A shows results of an experiment demonstrating the bacteriacapture efficiency of a microfluidic system designed in accordance withsome embodiments;

FIGS. 12B and 12C show results of an experiment demonstrating thebacteria capture efficiency of a microfluidic system at different flowrates and electric field settings in accordance with some embodiments;

FIGS. 13A and 13B show the results of capture efficiency experimentsperformed using a microfluidic device designed in accordance with someembodiments;

FIG. 14 shows capture efficiency results of an on-chip quantificationtechnique for captured bacteria in accordance with some embodiments;

FIG. 15 is a flow chart of a process for quantifying bacteria in asample in accordance with some embodiments;

FIG. 16 schematically illustrates a process for enriching bacteria in asample in accordance with some embodiments;

FIG. 17A illustrates a system configuration for enriching bacteria in asample in accordance with some embodiments;

FIG. 17B illustrates an alternate system configuration for enrichingbacteria in a sample in accordance with some embodiments;

FIGS. 18A-18D illustrate a process for non-specific (broad spectrum)capture of bacteria in a sample in accordance with some embodiments;

FIGS. 19A-19D illustrate a process for capturing a target bacterialspecies in a sample including multiple bacterial species in accordancewith some embodiments;

FIGS. 20A-20E illustrate a process for non-specific capture andselective release of bacteria in a sample in accordance with someembodiments;

FIGS. 21A and 21B show results of bacteria enrichment experiments inaccordance with some embodiments;

FIG. 22A schematically illustrates a system for sorting bacteria from acomplex sample including multiple bacterial species in accordance withsome embodiments;

FIG. 22B schematically illustrates a microfluidic device having fourelectrode systems for sorting bacteria from a complex sample inaccordance with some embodiments;

FIG. 22C illustrates cross-over frequencies for different bacterialspecies in a complex sample that may be used to sort bacteria inaccordance with some embodiments;

FIGS. 23A-23C illustrate a process for separating multiple bacterialspecies in a complex sample in accordance with some embodiments;

FIGS. 24A-24C illustrate a process for separating multiple bacterialspecies and other components in a complex sample in accordance with someembodiments;

FIGS. 25A-25C illustrate a process for separating live from deadbacterial species using multiple electrode systems in accordance withsome embodiments;

FIGS. 26A-26C illustrate a process for separating live from deadbacterial species using a single electrode system in accordance withsome embodiments;

FIGS. 27A-27E illustrate images of selective capture of bacterialspecies by tuning an electric field based on cross-over frequencies fordifferent bacterial species in accordance with some embodiments;

FIGS. 28A and 28B illustrate images of selective capture of bacterialspecies by tuning an electric field based on cross-over frequencies fordifferent bacterial species in accordance with some embodiments;

FIGS. 29A and 29B illustrate plots showing viability of bacteriafollowing capture and release using some embodiments;

FIG. 30 shows viability results for bacteria captured and released inaccordance with some embodiments;

FIG. 31 illustrates a schematic of a computing system that may be usedin accordance with some embodiments;

FIGS. 32A-32F illustrates capture of fungus, yeast, and bacteria,respectively in accordance with some embodiments;

FIG. 33 illustrates a circular assembly of electrodes that may be usedin accordance with some embodiments of the technology described herein;

FIG. 34 illustrates a sensor assembly that may be used in combinationwith the electrode assembly of FIG. 33, according to some embodiments;

FIG. 35 illustrates the sensor assembly of FIG. 34 fabricated on top ofthe circular electrode assembly of FIG. 33, according to someembodiments;

FIG. 36A illustrates the circular electrode assembly of FIG. 33fabricated on top of the sensor assembly of FIG. 34, according to someembodiments;

FIG. 36B illustrates an example device that includes supplementary wiresarranged to provide a field gradient in a region of a central sensinglayer, according to some embodiments;

FIG. 37A illustrates a layout for an example microfluidic device,according to some embodiments; and

FIGS. 37B-37H illustrate different geometries of electrodes for highsurface coverage to achieve high electric field gradients, according tosome embodiments.

DETAILED DESCRIPTION

Aspects of the technology described herein relate to an apparatus andmethods for detecting, separating, quantifying, and/or enrichingbiological organisms (e.g., bacteria) present in a fluid sample. Inparticular, the technology described herein provides techniques forrapid detection, separation, purification, and/or quantification ofbacteria in a sample using a microfluidic system comprising one or moreelectrodes configured to generate dielectrophoretic forces that act onthe sample.

Microbial (e.g., bacterial, viral and fungal) contamination is a seriousand growing global threat to human health and economic development. Anexample of a conventional technique to assess the presence and degree ofmicrobial contamination in a sample is the Plate Counting Method (PCM),which is shown schematically in FIG. 1. As shown, PCM typically includesat least four steps. In step 1, a sample to be analyzed is manuallyplaced in each of multiple test tubes, and the sample in each test tubeis diluted to a desired concentration using a buffer solution. In step2, the diluted samples are plated onto petri dishes containing agarmedia. Petri dishes including dilution media only (i.e., without thesample) are also plated for use as controls for comparison against theplated diluted samples. In step 3, the plurality of plated samples, thedilution media plates, and empty agar plates are cultured for 24 hoursto 14 days to enable microbial particles to grow on the media within thepetri dishes. In step 4, the number of bacterial colonies on each of theplates cultured in step 3 is determined, for example, using amicroscope.

PCM is routinely used in medical, pharmacological and food industries toidentify bacterial contamination. However, PCM is slow, only moderatelysensitive, labor intensive and prone to human errors. For instance, asshown, the entire PCM process takes 1-14 days, includes several manualsteps in which human intervention is needed, and requires a large numberof plated samples at different dilutions and controls. There istherefore a need for new technologies that allow for faster, moresensitive and more reliable assessment of microbial contamination.

Dielectrophoresis (DEP) has shown promise for particle separation;however, it has not yet been applied in clinical settings. For instance,only small sample volumes with unrealistically high bacterialconcentrations on the order of 10³-10⁷ CFU/mL have been processed, whichlimits the applicability of DEP microbial capture methods. DEP particleseparation has been achieved only to a limited extent and the separationis restricted to specific cell types, (e.g., separation of Escherichiacoli from Bacillus subtilis). Unfortunately, separation of small cells(˜1 μm in diameter, the size of many pathogenic bacteria) using DEP hasbeen notoriously difficult. For instance, small bacterial particlesundergo significant Brownian motion that adds a time dependent variationin their position, and thus the specificity of separation decreases forsmall cells, which has previously been thought to limit theapplicability of the DEP technique for detecting and/or separatingbacteria in a sample.

Some embodiments of the technology described herein relate to a novelDEP bacterial capture and separation technique (also referred to hereinas “Fluid-Screen” or “FS”) that addresses at least some of thelimitations of prior DEP techniques. As described in further detailbelow, the efficiency of bacterial capture using the techniquesdescribed herein are measured and compared to bacterial capture usingthe standard PCM technique.

Although capture and separation of bacteria from a sample is describedherein, it should be appreciated that biological particles other thanbacteria, for example, different cells, yeast, mold, fungus, viruses,etc. can also be detected, quantified, separated, and/or enriched usingone or more of the techniques described herein. Indeed, the technologydescribed herein has been shown to effectively capture, detect,quantify, and separate a wide range of diverse microorganisms including,but not limited to, both Gram (−) and Gram (+) bacteria, multiplebacterial morphologies, both individual bacteria and cell aggregates,yeasts or molds (including conidia, conidiophores and hyphae), andviruses. Table 1 below illustrates a summary of some microorganisms thathave been successfully captured and detected using the techniquesdescribed herein.

TABLE 1 Example microorganisms detected using the techniques describedherein Microorganism Media Media PBS PBS Microfluidic Differentiation[Dilution] pH [Dilution] pH Chip Type Stain E. coli Gram (−) CHO, 1:1007.09 1:1000 7.4 Static Sybr Green B. subtilis Gram (+) CHO, 1:100 7.091:1000 7.4 Static Sybr Green P. aeruginosa Gram (−) CHO, 1:100 7.091:1000 7.4 Static Sybr Green S. aureus Gram (+) CHO, 1:100 7.09 1:10007.4 Static Sybr Green C. albicans Yeast CHO, 1:100 7.09 1:1000 7.4Static Sybr Green A. brosiliensis Mold CHO, 1:100 7.09 1:1000 7.4 StaticN/A

A microfluidic system designed in accordance with some embodiments mayalso be used to capture yeast, mold, and bacteria in a Chinese hamsterovary (CHO) cell matrix, examples of which are shown in FIGS. 32A-E.FIGS. 32A and 32B show the response of the fungus Aspergillus niger whenthe electric field is off (FIG. 32A) resulting in no capture and whenthe electric field is on (FIG. 32B) resulting in capture on the edges ofthe electrode. FIGS. 32C and 32D show the response of the yeast C.albicans when the electric field is off (FIG. 32C) resulting in nocapture and when the electric field is on (FIG. 32D) resulting incapture on the edges of the electrode. FIGS. 32E and 32F show theresponse of the bacteria S. aureus when the electric field is off (FIG.32E) resulting in no capture and when the electric field is on (FIG.32F) resulting in capture on the edges of the electrode.

In accordance with some embodiments, a fluid sample containing bacteriais processed in a microfluidic system. For example, in a microfluidicdevice, the sample may be subjected to DEP forces and/or electroosmosis(EO) to enable detection, separation, purification and/or quantificationof bacterial particles in the fluid sample. Examples of a microfluidicsystem suitable for use in accordance with the techniques describedherein, include the Fluid-Screen Microfluidic System, aspects of whichare described in U.S. patent application Ser. No. 16/093,883 and titled“ANALYTE DETECTION METHODS AND APPARATUS USING DIELECTROPHORESIS ANDELECTROOSMOSIS,” filed on Oct. 15, 2018, and U.S. patent applicationSer. No. 14/582,525 and titled “APPARATUS FOR PATHOGEN DETECTION” filedon Dec. 24, 2014, each of which is hereby incorporated by reference inits entirety.

FIG. 2 illustrates an example system for detecting bacteria in a sample,in accordance with some embodiments. As shown in FIG. 2, the system 200comprises a microfluidic device 204 in communication with a computingdevice 210.

The microfluidic device 204 may be any suitable device, examples ofwhich are provided herein, in particular, with respect to FIG. 3. Insome embodiments, microfluidic device 204 comprises a microfluidic chiphaving one or more passages (e.g., microfluidic channels or chambers)through which a fluid sample 202 is provided for analysis. Although theterm “microfluidic channel” or simply “channel” is used herein todescribe a passage through which fluid flows through microfluidic device204, it should be appreciated that a fluid passage having any suitabledimensions may be used as said channel, and embodiments are not limitedin this respect. Microfluidic device 204 may comprise a single channelor multiple channels configured to receive a single sample 202 (e.g., toperform different analyses on the sample) or multiple channelsconfigured to receive different samples for analysis. In embodimentshaving multiple channels, the microfluidic device may be configured toprocess the single sample or multiple samples in parallel (e.g., at thesame or substantially the same time).

As described herein, sample 202 may include any fluid containingbacteria or other microorganism of interest. In some embodiments, thesample comprises a biological fluid such as saliva, urine, blood, water,any other fluid such as an environmental sample or potentiallycontaminated fluid, protein matrices, mammalian cell culture, bacterialculture, growth media, active pharmaceutical ingredients, enzymeproducts, or substances used in biomanufacturing, etc.

As shown, microfluidic device 204 includes at least one electrode 206.The at least one electrode 206 may be configured to receive one or morevoltages to generate positive and/or negative dielectrophoresis (DEP)force(s) that act on a sample arranged proximate to the at least oneelectrode. In some embodiments, the at least one electrode 206 may beconfigured to receive one or more voltages (e.g., one or more ACvoltages) to generate at least one dielectrophoresis force orelectroosmotic (EO) force that acts on the sample. The at least one DEPand/or EO force may cause certain components of the sample to moverelative to (e.g., be attracted to or repulsed from) a surface of the atleast one electrode 206. For example, in the absence of an electricfield, bacteria and other components of the sample 202 may move freelyrelative to the surface of the electrode. In the presence of theelectric field at least some components (e.g., bacteria) in the samplemay be attracted to the electrode surface.

The small size of bacteria presents an obstacle to optical observationand quantification of bacteria in the sample. The inventors haverecognized that activation of the at least one electrode 206 results inan electric field that may be used to selectively trap bacteria on thesurface of the electrode(s). When used with an optical detection system,capturing bacteria on the surface of the electrode(s) may prevent thebacteria from moving in and out of focus of the optical device to enablereal-time bacteria detection and quantification, a process referred toherein as “on-chip quantification.”

The electric field used to capture the bacteria concentrates thebacteria, which enables imaging with fluorescence microcopy or anotheroptical detection technique. Accordingly, bacterial capture using thetechniques described herein allows for detection and quantification ofbacteria at significantly lower limits compared to some conventionalmethods, such as the PCM technique described in connection with FIG. 1.The ability to detect and/or quantify bacteria in a sample, even insmall amounts, may be useful in applications including, but not limitedto, biomanufacturing, gene therapy, analysis of patient samples, vaccinedevelopment and/or biothreat detection.

For example, the at least one DEP and/or EO forces acting on the samplemay cause bacteria to separate from other components of the sample(e.g., via positive DEP). Bacteria in the sample may be attracted to thesurface of the at least one electrode 206 allowing for enhanceddetection and/or quantification, despite the small size and/or smallamount of the bacteria in the sample. Although, microfluidic device 204is illustrated as having a single electrode, it should be understoodthat in some embodiments, microfluidic device 204 comprises multipleelectrodes arranged in any suitable configuration. The at least oneelectrode(s) 206 may have any suitable shape. Non-limiting examples ofelectrode shapes and designs that may be used in accordance with someembodiments are further described below in connection with FIGS. 7A-7Fand FIGS. 33-37.

System 200 may further comprise a computing device 210 configured tocontrol one or more aspects of microfluidic device 204. For example,computing device 210 may be configured to direct the sample 202 into achannel of the microfluidic device. In some embodiments, computingdevice 210 is configured to control the at least one electrode 206 togenerate the at least one DEP force and/or EO force acting on the sample202. In some embodiments, computing device 210 may cause one or morecomponents of the microfluidic system (e.g., an optical device) toperform one or more of detection, quantification, separation, and/orpurification of the bacteria or other microorganisms in the sample.Non-limiting examples of a computing device 210 that may be used inaccordance with some embodiments are further described herein, forexample, with respect to FIG. 31.

An example microfluidic device configured to process a sample inaccordance with the techniques described herein is shown in FIG. 3,which is reproduced from U.S. patent application Ser. No. 13/664,967,now U.S. Pat. No. 9,120,105, entitled “ELECTRONIC DEVICE FOR PATHOGENDETECTION” filed on Oct. 31, 2012, which is hereby incorporated byreference in its entirety. Device 10 in FIG. 3 comprises a samplechamber 12 and a chamber 14 containing a reference solution which may insome embodiments include a separator which purifies the referencesolution from contaminants. In some embodiments, device 10 may notinclude the chamber containing the reference solution.

Chambers 12 and 14 are connected by micropumps adapted to force eitherfluid around the passage 18 and through separator passage 16. First, thesample comprising bacteria and other components may be pumped throughthe separator. The separator includes one or more electrodes configuredto apply a dielectrophoretic, electroosmotic, and/or other AC kineticforce on the components of the sample, which results in bacteria in thesample being selectively attracted toward the bottom of the figure. Theother components not attracted toward the electrode(s) may be trapped inchamber 22, while the bacteria are drawn into the holding chamber 24 byconcentrator 20, which the separator and the condenser may in someembodiments comprise a set of coaxial interdigitating rings or archeshaving independent voltages. Once the bacteria are held by theconcentrator 20, the buffer solution may be pumped from chamber 12around the bend 18 and through the separator passage 16 to flush thechamber 24, effectively changing the medium in which the bacteria arefound and eliminating any residual unfiltered elements. The bacteria canthen be released from concentrator 20 (by removing the electric field)and may be drawn towards analyzer array 26 (which itself may be providedwith one or more electrodes adapted to attract the bacteria thereto).

Device 10 uses dielectrophoresis for purposes of separating bacteriafrom other components of a sample. Dielectrophoresis uses a natural orinduced dipole to cause a net force on a particle in a region having anelectric field gradient.F=2πε_(m) R ³Re[ CM (ω)·∇ E ²(r,ω)]

This force depends on the Clausius-Mossotti factor CM(w) defined by

${CM}{(\omega) = \frac{\epsilon_{p}^{o} - \epsilon_{m}^{o}}{\epsilon_{p}^{o} + {2\epsilon_{m}^{o}}}}$where ∈^(o) is the complex permittivity,

$\epsilon^{o} = {\frac{\sigma}{i\;\omega}.}$

In some embodiments, the values for σ and ω are chosen to reach amaximal separation force between the bacteria or other analyte to beseparated and other components in the solution being processed by thedevice. This can be accomplished by compiling knowledge concerning boththe bacteria and other components to be separated. The differentialresponse of the bacteria and other components of a sample to an appliedelectric field can be inspected for its extrema which will show thegreatest differential response tending to separate the bacteria from theother components. The frequency of the applied AC voltage used forseparation may be chosen, while the conductivity of the solution can becontrolled by titration of a known amount of solution of knownconductivity (or equivalently, salinity). Alternatively, a feedbacktechnique may be used by measuring the conductivity of the solution andadding saline or deionized water (for instance) until a desiredconductivity is reached. A reference measurement may be used for qualitycontrol and identification of the solution. A differential measurementof the control signal (no contamination) with an actual signal (withlabeled contaminants) may be used. Conductivity and complex permittivitymeasurements may be implemented at multiple stages in the devices forquality control of fluid mixing and feedback adjusting the mixing rate.As will be appreciated by one skilled in the art, such analysis of adifferential response may be performed for any pair of species inquestion in a given sample.

FIG. 4 illustrates an example system 400 for detecting the presence ofbacteria in a sample, in accordance with some embodiments. System 400includes microfluidic device 408 (e.g., a microfluidic chip) thatincludes one or more electrodes for generating DEP and/or EO forces thatact on a sample 404 provided as input to the system. Sample 404 maycontain bacteria for which detection, separation, purification, and/orquantification may be performed. The one or more electrodes may bearranged in any suitable configuration within the microfluidic device408. For instance, in embodiments that include multiple electrodes theelectrodes may be arranged in one-dimension along the flow direction ofthe fluid, perpendicular to fluid flow direction or on a diagonalrelative to the fluid flow direction. In some embodiments, amultidimensional (e.g., 2-dimensional, 3-dimensional) array ofelectrodes may be used. For instance, a dense array of electrodesarranged both along the direction of fluid flow and perpendicular to thedirection of fluid flow may be used.

As shown in FIG. 4, a flow system 402 is provided. The flow system 402may provide a solution for transporting the sample 404 to themicrofluidic device 408. A first pump 406 may be used to pump thesolution and the sample 404 to the microfluidic device 408 at apredetermined flow rate. First pump 406 may be of any suitable type. Insome embodiments, as further described herein, first pump 406 isomitted, and sample 404 is manually loaded (e.g., using a pipette) asinput to one or more channels of microfluidic device 408.

Microfluidic device 408 is configured to receive sample 404 forprocessing. Microfluidic device 408 may include one or more channelsthrough which the sample 404 flows. The one or more channels may includeat least one electrode formed therein or adjacent thereto. For instance,the at least one electrode may be formed within a channel. The at leastone electrode, when activated, is configured to generate an electricfield that acts on the sample 404 as it flows through the one or morechannels. An electrical system 412 (e.g., a signal generator orcontroller) is configured to provide one or more voltages to the atleast one electrode of the microfluidic device 408 to tune theproperties of the electric field for capture of a particularmicroorganism or microorganisms of interest. Further aspects of theelectrical system 412, including example protocols for operating themicrofluidic device 408 are provided herein.

An optical system 410 may be provided to facilitate analysis of thesample 404 by performing on-chip quantification. For example, theoptical system 410 may comprise one or more optical sensors for viewingand/or imaging the sample. The optical sensor(s) may provide forenhanced detection and/or quantification of the bacteria and/or theother components of the sample 404 relative to detection andquantification techniques that require separate culturing of capturedbacteria or an effluent sample from the device. Any suitable opticaldetector may be used. In some embodiments, the optical sensor(s)comprises a digital camera. In some embodiments, the optical sensor(s)comprises electronic sensors including CMOS compatible technology. Insome embodiments, the optical sensor(s) comprise fiber optics. However,any suitable optical sensor(s) may be used. In some embodiments,bacteria in the sample are stained with a fluorescent dye and theoptical system 410 is configured to perform fluorescence microscopy ofcaptured stained bacteria. In some embodiments, optical system 410 isconfigured to capture one or more images of the at least one electrodewhile the sample is flowing through the microfluidic device 408. In someembodiments, the detector comprises nanowire and/or nanoribbon sensors.

System 400 also includes computer 430 configured to control an operationof optical system 410 and/or to receive images from optical system 410and to perform processing on the received images (e.g., to count anumber of bacteria trapped by the microfluidic device 408). In someembodiments, the received images are analyzed to determine the number ofbacteria captured by the at least one electrode. For instance, bacteriamay be identified in the received images as spots (e.g., fluorescentspots) located on the edges of the electrodes. In this way a capturedtarget bacterial species may be differentiated from other components inthe sample that are not captured and may appear as floating above the atleast one electrode or located between electrodes, examples of which areshown and described below in connection with FIGS. 27A-E and 28A-B.

After the sample 404 is processed by the microfluidic device 408 and/oroptical system 410 to capture and/or quantify bacteria on theelectrode(s), the sample 404 may be removed from the microfluidic device408. For example, a second pump 416 may be provided for pumping thesample 404 out of the microfluidic device 408. The second pump 416 maybe of any suitable type. In some embodiments, system 400 comprises aflow sensor 414 for measuring a flow rate at which the sample 404 isremoved from the microfluidic device 408. The flow sensor 414 and thesecond pump 416 may be in communication to control a flow rate at whichthe sample 404 is removed from the microfluidic device 408.

As described herein, system 400 may be used for separating bacteria fromother components in sample 404. System 400 comprises a waste region 418arranged to receive other components of the sample 404 which have beenseparated from the bacteria by the microfluidic device 408 andsubsequently removed from the sample 404, for example, using the secondpump 416. In the description below, analysis of the fluid collected inwaste region 418 may be referred to as analysis of the “effluentsample.” System 400 may further include effluent region 420 forreceiving a purified version of sample 404 containing substantially onlytarget bacteria that were captured using microfluidic device 408.

In some embodiments, an amount of time needed to process a sample usingsystem 400 is substantially less than an amount of time required toprocess a sample using a conventional sample processing system (e.g.,PCM shown in FIG. 1). As shown in FIG. 4, processing a sample usingsystem 400 may include three steps. In step 450, a sample is provided asinput to microfluidic system 408 and bacteria are captured from thesample in the presence of an applied electric field. In step 460,automated on-chip quantification is performed, for example, using anoptical system and computer 430 to analyze one or more images recordedby optical system 410. In step 470, further analysis may be performed onwaste 418 and/or effluent sample 420, as desired. In sum, the entireprocess for detecting and/or quantifying bacteria in a sample usingsystem 400 may take on the order of minutes or an hour to a few hours,which is substantially faster than the multiple days (e.g., 1 to 14days) typically required to process samples using PCM discussed withreference to the system in FIG. 1.

In some embodiments, rather than pumping sample 404 through one or morechannels through which the sample flows, sample 404 may be manuallyprovided as input to microfluidic device 408 for analysis. For instance,one or more droplets of sample 404 may be provided as input tomicrofluidic device 508 using a pipette or other suitable technique. Insuch embodiments, the sample is analyzed in a “static” condition ratherthan in a condition in which bacteria are captured by the at least oneelectrode as the sample flows past the electrode(s) (e.g., as in thecase of system 400 as shown in FIG. 4). FIG. 5 illustrates a system 500for detecting bacteria in a sample, according to some embodiments. Asshown, system 500 may include many of the same components as system 400,but may omit certain components of the system 500, such as the firstpump 406, which are not needed when the sample is manually provided asinput to the microfluidic device.

FIG. 6A illustrates a schematic diagram of capturing bacteria from asample using system 500. As shown, in the presence of an electric field605 generated using electrode 620, bacteria 610 are attracted to theelectrode 620 by a positive DEP force acting on the bacteria 610 in thesample. The trapped bacteria can then be imaged using optical system 410to perform direct on-chip quantification. In some embodiments, a systemwith multiple electrodes may be used and the sample may be provided forinterrogation by each of the electrodes.

FIG. 6B illustrates a schematic diagram of capturing bacteria from asample using system 400. In particular, the sample may be introduced tothe microfluidic device from an influent region 630. Bacteria 610included in the sample may be attracted to the surface of electrode 620in the presence of an electric field 605 generated by the electrode asthe sample flows past the electrode in the microfluidic device at apredetermined flow rate. Components not captured by the electrode 620may be provided into an effluent region 640 (e.g., waste region 418 inFIG. 4) for further analysis or to be discarded. Additionally, oralternatively, bacteria captured by the electrode 620 may be released toeffluent region 640 (or a different effluent region) by adjusting theapplied electric field (e.g., by deactivating the electrode 620 therebyturning the electric field off).

FIGS. 7A-7F illustrate schematics of an electrode design that may beused in accordance with some embodiments. As shown in FIG. 7A, theelectrode design may be a system of concentric rings or arcs in whichevery other ring from the center is electrically connected. Thealternating rings may be connected to a voltage source having differentpolarities, thereby creating electric field gradients used to capturebacteria on the surface of the electrode. FIG. 7B shows a schematic of apart of an electrode system with applied alternating voltage polarityfor the ring structure that may be used in some embodiments. FIG. 7Cshows a zoomed-in schematic of part of the electrode design shown inFIG. 7B. For visual clarity the electrode is shown black, and glass,which is between electrodes, is shown in gray. FIG. 7D shows fluorescentimaging of the electrode when the electric field is off and FIG. 7Eshows fluorescent imaging of the electrode when the electric field ison. A comparison of FIGS. 7D and 7E shows that E. coli bacteria (dots)are captured on the electrode edges of the ring structure when a voltagehaving an amplitude of 10 V and a frequency of 10 MHz is applied to theelectrode. FIG. 7F shows an overview of the electrode with GFP-labelledbacteria captured from a 0.001×PBS solution spiked with bacteria. Thedevice does not show saturation, even with high levels of bacteria.

In some embodiments, for example, the at least one electrode comprisesat least one circular-shaped and/or partially-center-symmetric electrode(e.g., the electrode design shown in FIG. 7A). Additional exampleelectrode designs are provided in FIGS. 33-37H.

For example, some embodiments make use of a circular assembly of coaxialor spiral-shaped electrodes such as shown in FIG. 33, where two or moreindependent voltages may be applied to the odd and even rings. Thisallows for an electric field gradient to be created in the regionbetween the rings. The assembly of electrodes is constructed in such away as to maximize the effects of the electric field on controlling themotion of the sample components.

Such a device may be used to draw components of a sample, e.g., bacteriaor other elements to the sensor array, which may be composed of elementssuch as those shown in FIG. 34, namely source 3401 and drain 3402,nanowire, nanoribbon or active sensing layer 3405, silicon or othersemiconducting substrate 3404 and SiO₂ or other insulating interlayer3403.

The sensor assembly of FIG. 34 may be fabricated on top of circular DEPelectrodes as shown in FIG. 35, or a set of circular electrodes may befabricated on top of (or underneath, in some embodiments) the SiO₂ orother insulating layer as shown in FIG. 36A. Alternatively, twosupplementary wires 3606 may be used as shown in FIG. 36B to provide afield gradient in the region of the central sensing layer.

A further aspect allows for selective treatment of individual sensors ina sensor array, such that each sensor or group of sensors can be madesensitive to a particular pathogen or family of pathogens. The sensorarray may be such as that disclosed in U.S. patent application Ser. No.12/517,230 titled “CMOS-COMPATIBLE SILICON NANO-WIRE SENSORS WITHBIOCHEMICAL AND CELLULAR INTERFACES” filed on Jul. 12, 2010, which ishereby incorporated by reference in its entirety. In some embodiments,the wires of the array form the bases of field-effect transistors, andthus implement nanowire FETs or FETs.

FIG. 37A shows the layout of a microfluidic device in accordance withsome embodiments. FIGS. 37B-H illustrate different geometries ofelectrodes for high surface coverage to achieve high electric fieldgradients in accordance with some embodiments. In some embodiments, anelectrode having one of the geometries shown in FIGS. 37A-H may coverthe entire surface of a chamber (e.g., wall, top and/or bottom) of afluidic device, examples of which are discussed above. The electrodesinduce high field gradients, so that samples introduced into the chamberare exposed to high electric fields regardless of their position in thechamber. Such electrode design with a high surface coverage allows forcontrol of over 99% of bacteria present in the sample and reduces falsenegatives.

As described herein, a further aspect in accordance with someembodiments involves the use of electroosmosis in addition todielectrophoresis for transport. The frequencies at which electroosmosisare effective (e.g. tens of kHz) are widely separated from those usefulin DEP, and therefore the two methods can be used simultaneously toprovide a larger variety of separation regimes, and for a wider varietyof objects to be separated.

In some embodiments, a high-density gradient of electric field isinduced by electrodes which are matched to bacteria size, so thatbacteria particles are within 10-500 times the size of the electrodeand/or electrode spacing.

FIG. 8 illustrates a process 800 for assessing variability inquantifying bacteria using the static system 500 shown in FIG. 5. In act810 a stock sample including bacteria is prepared. For instance,cultured bacteria (e.g., E. coli bacteria) may be collected from abiofilm area of one or more agar plates using a sterile inoculation loopand the collected bacteria may be suspended in an amount (e.g., 2 mL) ofPhosphate Buffered Saline (PBS) 1:1000 or another suitable buffersolution. The stock sample may be set up at a concentration (10⁷-10⁸ mL)using an optical density meter at 600 nm. An amount (e.g., 5 mL) of thestock sample may be prepared and a small amount (e.g., 1 mL) of thesample may be reserved for plating in serial 10× dilutions (e.g., 10⁻⁴,10⁻⁵ and 10⁻⁶) on Tryptic Soy Agar (TSA) agar or selective agar media(MacConkey (MAC)) as a control after stock sample preparation. The stocksample prepared in act 810 is then used as a base to prepare the samplesto be processed using system 500 to detect bacteria in the samples.

Process 800 then proceeds to act 812, where one or more sample dilutionsare created. Fluorescent dye is added to the dilutions to facilitateimaging with the optical system. For instance, a concentration ofbacteria in the test sample may be set at 100-400 CFU/mL in PBS dilutedto 1:1000 by serial 10× dilution of the stock sample. To visualize thebacterial response to the applied electric field, a small amount (1 μL)of fluorescent dye Sybr Green (or another fluorescent dye) is added to 1mL of the test sample and the solution is incubated (e.g., for 15 min atroom temperature in darkness).

Process 800 then proceeds to act 814, where the diluted and stainedsample is loaded into the chip. For instance, a micropipette may be usedto load 2 μL of the stained sample into a channel of the chip. Process800 then proceeds to act 816, where parameters for the applied electricfield are determined and the electrode(s) in the microfluidic device areactivated, resulting in the capture of bacteria by the electrode(s) bythe applied DEP forces acting on the bacteria. Process 800 then proceedsto act 818, where the bacteria captured by the electrode in the presenceof the applied electric field are quantified using fluorescencemicroscopy of the chip. After removing the electric field and flushingthe chip with a solution to remove any bacteria microfluidic device,acts 814-818 may be repeated with a new diluted andfluorescently-labeled sample to generate multiple repeats of the on-chipquantification measurement. The chip may be imaged between each repeatto verify chip cleanliness. In the results described herein inconnection with FIGS. 9 and 10A-C, multiple (e.g., 12) repeats weregenerated to assess variability in bacterial quantification. A nullcontrol image of the electrode without bacteria using the samefluorescent application was also collected for comparison.

Two biological samples including bacteria were processed using process800. Twelve technical repeats were performed to demonstrate theprecision and repeatability of bacteria quantification with the system500. FIG. 9 shows bar plots of the total number of bacteria countedacross twelve repeats using process 800 for a first biological sampleand a second biological sample, respectively. Each vertical barrepresents the number of bacteria counted for an individual experiment.A variability range of +/−0.5 log is shown as a shaded horizontal bar inFIG. 9. As shown, the bacterial quantification variability acrossrepeats using the system 500 was substantially smaller than the +/−0.5log range for variability (which is a range often used to validate newmethods for clinical use), thereby demonstrating a system for bacterialcapture and quantification with improved precision and repeatabilitycompared to conventional techniques such as PCM, which typically havemeasurements at or near the boundaries of the acceptable +/−0.5 logspread.

The precision and repeatability in enumeration of multiple microfluidicchips designed in accordance with the techniques herein is shown inFIGS. 10A-C. Eight different microfluidic chips were used to quantifythe number of captured bacteria across the two biological samples. Eachchip was used to count the bacteria three times (each chip had threechannels), demonstrating a high degree of precision and repeatability inchip capture and image processing performance. FIGS. 10A and 10B showthe total number of bacteria counted on each of four different chips forthe first and second biological samples, respectively. For reference, aswith the bar plot in FIG. 9, the +/−0.5 log variability range is shownas a shaded horizontal bar in FIGS. 10A-C. The variability from chip tochip is substantially smaller than the +/−0.5 log variability spread foreach of the biological samples. FIG. 10C shows a statistical comparisonof the two biological samples across twelve repeats for each sample.Overall, there was no significant difference between biological repeats,demonstrating the very high precision and repeatability in the captureand quantification of bacteria using the techniques described herein.Statistical analysis from the eight different chips tested is summarizedin Table 2 below.

TABLE 2 Statistical analysis of the repeatability of bacteria capturecompared between eight microfluidic chips. True number of bacteria MeanSD % CV Biological repeat 1 Chip Channel 1 350 348.000 23.065 6.628 1Channel 2 324 Channel 3 370 Chip Channel 1 279 302.333 45.709 15.119 2Channel 2 355 Channel 3 273 Chip Channel 1 297 283.667 15.275 5.385 3Channel 2 267 Channel 3 287 Chip Channel 1 287 4 Channel 2 228 252.00031.000 12.302 Channel 3 241 Biological repeat 2 Chip Channel 1 254263.333 11.372 4.319 1 Channel 2 260 Channel 3 276 Chip Channel 1 235251.000 21.932 8.739 2 Channel 2 242 Channel 3 276 Chip Channel 1 312269.333 36.950 13.719 3 Channel 2 248 Channel 3 248 Chip Channel 1 239297.333 70.727 23.787 4

FIG. 11 illustrates a process 1100 for assessing the efficiency ofbacterial capture using the microfluidic system 400 shown in FIG. 4. Inact 1112, an influent sample is connected to an input pump of themicrofluidic system. A small amount (e.g., 1 mL) of influent sample isreserved for culturing using PCM, as discussed in further detail below.Process 1110 then proceeds to act 1114, where a desired flow rate andelectric field parameters are selected. As discussed further below,capture efficiency of system 400 at a plurality of flow rates wastested. The electric field parameters may be tuned to attract multipletypes of bacteria to the electrode(s) within the microfluidic system ormay be tuned to selectively capture one or more types of bacterialspecies while repelling one or more other types of bacterial species. Insome embodiments, setting electric field parameters comprises setting anamplitude and/or frequency of a voltage provided to activate the one ormore electrodes within the microfluidic system.

Process 1100 then proceeds to act 1116, where the electric field isturned on in accordance with the selected parameters and the influentsample is pumped at the selected flow rate through one or more channelsin the microfluidic device associated with the one or more electrodes.As the sample traverses the portion of the channel(s) proximate to theone or more electrodes, bacteria are captured from the sample on thesurface of the electrode(s) due to a positive DEP force acting on thebacteria in the sample. The remaining components in the sample notcaptured by the electrode proceed through the channel(s), where they arecollected as effluent in act 1118. In some embodiments, the componentsin the sample that are not captured by the electrode(s) are first storedin a chamber prior to being pumped out of the microfluidic system intoan effluent region for further analysis.

Process 1100 then proceeds to act 1120, where the capture efficiency ofthe microfluidic device is determined. Capture efficiency may bedetermined using one of at least two techniques. In a first technique,“on-chip quantification” of bacteria captured by the electrode isperformed to count the number of captured bacteria in one or more imagescaptured by an optical system. For instance, following capture of thebacteria by the electrode(s), an optical system may be used to captureone or more images of the electrode(s) while the bacteria are capturedby the electrode. The number of bacteria captured on the electrode(s)may be then be quantified by analyzing the one or more images capturedby the optical system and compared to an analysis of the effluentsample.

In a second technique, “PCM quantification” of bacteria is performed bycomparing an amount of bacteria in the influent sample with an amount ofbacteria in the effluent sample. For instance, PCM quantification mayproceed according to steps described in connection with FIG. 1.

FIGS. 1 and 4, respectively show general schematics of the PCMquantification and on-chip quantification counting techniques forassessing efficiency of bacterial capture of a microfluidic systemdesigned in accordance with some embodiments. For both techniques 1 mLof the effluent (output sample) was collected and plated immediately onMAC agar plates for enumeration using PCM to calculate the number ofColony Forming Units (CFUs).

For PCM quantification, a biological sample provided as input to themicrofluidic device is referred to as the influent sample. The samplethat exits the chip following collection of the bacteria on theelectrode(s) in the microfluidic device is collected as the effluentsample. Both the influent sample and the effluent sample are cultured onproper media, and after 24 hours of growth, the number of bacteria arequantified, and the two numbers are compared. The factor that shows theefficiency of the process is called the capture efficiency and iscalculated as:

${Capture}\mspace{14mu}{Efficiency}{= {\left( {1 - \frac{Conc_{eff}}{Conc_{\inf}}} \right)*100\%}}$where Conc_(eff) is the concentration of bacteria in the effluent sampleand Conc_(inf) is the concentration of bacteria in the influent sample.

FIG. 12A illustrates the capture efficiency for various influentconcentrations of bacteria as determined using PCM quantification. Asshown, the capture efficiency is 100% or nearly 100% at all influentconcentrations tested. FIG. 12B illustrates capture efficiency forvarious influent concentrations and flow rates. As shown, the captureefficiency depends on flow rate and electric field settings (e.g., howstrong the DEP force is for particular bacteria being captured). At flowrates less than 480 uL/min the capture efficiency is greater than orequal to 99.99%. Though not shown, flow rates as low as 10 uL/min weretested and showed capture efficiencies of at least 99.99%. As flow ratesare increased, the capture efficiency is decreased, but still remainsabove 99.9% in experiments in which the flow rate was 960 uL/min orless. FIG. 12C shows additional capture efficiency results when theelectric field settings were changed from those used in the experimentthat produced the results in FIG. 12B. The results in FIG. 12C confirmthat capture efficiency depends on flow rate and electric fieldparameters.

Additional experiments in which an observed overall 100% bacterialcapture efficiency, as verified by PCM quantification, were alsoperformed. An unstained bacteria capture experiment was repeated in fourbiological replicates (each corresponding to a new separately grownbacterial sample) with three technical replicates (each being atriplicate repetition of the bacteria capture experiment, donesequentially, from the same biological replicate) per each biologicalreplicate for a total of 12 tests. All four biological repeats using thesystem 400 in each of the 12 total conducted experiments, demonstrated100% bacteria capture efficiency and repeatability. Detailed data of theadditional PCM quantification experiments is summarized in Table 3,including the number of bacterial colonies in the negative control,bacterial concentration in influent, bacterial concentration in effluentand the calculated capture efficiency. The number of CFUs in eachinfluent was between 20 CFU/mL and 420 CFU/mL.

TABLE 3 Bacterial capture results using PCM quantification and on-chipquantification techniques Plate-Count Method [cfu/mL] “PCMquantification” Biol Rep 1 Biol Rep 2 Biol Rep 3 Tech Rep 1 Tech Rep 2Tech Rep 3 Tech Rep 1 Tech Rep 2 Tech Rep 3 Tech Rep 1 Tech Rep 2 TechRep 3 [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Avecfu/ [Ave cfu/ [Ave cfu/ mL] mL] mL] mL] mL] mL] mL] mL] mL] Neg_(CTRL)0 0 0 0 0 0 0 0 0 Influent 7 × 10¹ 2 × 10¹ 1.7 × 10¹ 5.2 × 10¹ 1.2 × 10¹4.2 × 10¹ 6.5 × 10¹ 5.33 × 10¹ 2.0 × 10¹ ≤250 cfu/mL Effluent 0 0 0 0 00 0 0 0 Cap_(eff) [%] 100 100 100 100 100 100 100 100 100 On-ChipQuantification Neg_(CTRL) 6 15 3 4 3 12 26 13 5 Capture 184 130 198 124179 189 124 128 139 Total 178 115 195 120 176 177 98 115 134 Capture[Cap- Neg_(CTRL)]

Note that in the PCM quantification experiment having results summarizedin Table 3 and resulting in 100% capture efficiency, bacteria in theinfluent sample were not stained with any fluorescent stain. Lack ofbacterial staining in a PCM quantification experiment avoids anypotential growth inhibition by the fluorescent dye on MAC agar plates.For all conducted experiments, acceptable growth and viability range+/−0.5 log were reported.

FIGS. 13A and 13B show the results of capture efficiency experimentsperformed using a microfluidic device designed in accordance with thetechniques described herein, and using PCM quantification of bacteria ininfluent and effluent samples. FIG. 13A shows data presented as a meanand +/−SD from 3 technical replicates for each biological replicate.FIG. 13B shows data presented as a mean and +/−SD from four biologicalreplicates and their technical replicates, for a total of 12 tests. Theshaded bar represents a growth and viability variance range of +/−0.5log. In every experiment, the microfluidic device captured 100% ofbacteria, as evidenced by zero PCM growth in effluent samples. The 100%capture efficiency is maintained in a broad range of bacteriaconcentrations. Numeric values associated with the plots shown in FIGS.13A and 13B is shown in Table 4 below.

TABLE 4 Results of capture efficiency experiments according to someembodiments Plate-Count Method [cfu/mL] Biological Replicate 1Biological Replicate 2 Biological Replicate 3 Tech Rep 1 Tech Rep 2 TechRep 3 Tech Rep 1 Tech Rep 2 Tech Rep 3 Tech Rep 1 Tech Rep 2 Tech Rep 3[Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/ [Ave cfu/[Ave cfu/ [Ave cfu/ mL] mL] mL] mL] mL] mL] mL] mL] mL] Neg_(CTRL) 0 0 00 0 0 0 0 0 Influent 3.3 × 10² 2.4 × 10² 2 × 10² 2.8 × 10² 4.2 × 10² 1.3× 10² 1.5 × 10² 2.9 × 10² 0 × 10² ≤250 cfu/mL Effluent 0 0 0 0 0 0 0 0 0Cap_(eff) [%] 100 100 100 100 100 100 100 100 100 Biological Replicate 4Tech Rep 1 Tech Rep 2 Tech Rep 3 [Ave cfu/ [Ave cfu/ [Ave cfu/ mL] mL]mL] Neg_(CTRL) 0 0 0 Influent 1.9 × 10² 1.5 × 10¹ 2 × 10¹ ≤250 cfu/mLEffluent 0 0 0 Cap_(eff) [%] 100 100 100

Rather than comparing the number of cultured bacteria in influent andeffluent samples using PCM quantification as described above, someembodiments use “on chip quantification” of bacteria imaged while thebacteria are captured on the microfluidic chip. For on-chipquantification, the number of bacteria was determined by backgroundsubtraction from the total count on the chip. The total number ofbacteria was determined optically as a number of spots that produced afluorescent signal. A spot was counted if the size of the spotcorresponded to at least 25% of bacterial size, in this case more than 8connected pixels, which corresponds to 0.5 μm. First, bacteria wererecognized from the fluorescent images based on spot size and thedifference of intensity between the intensity maximum and thebackground. Electrode position was determined from the optical electrodeimage. Capture efficiency was calculated as: 1−Number of bacteriacaptured on electrode/Total number of bacteria

The fluorescent images were filtered with a digital bandpass filter topass the wavelength of the fluorophore (509 nm for green fluorescentprotein (GFP)); the range between black (minimum) and the brightestbacterium pixel (maximum) was then reassigned to a full color scale.

As shown in FIG. 14, direct “on chip quantification” of capturedbacteria may be a more reliable technique for quantifying bacteria in asample compared to a standard established, indirect “PCMquantification,” which requires converting the real number of capturedbacteria to CFU/mL values. Additionally, FIG. 14 shows that the direct“on chip quantification” technique yields a very small bacteria countingerror, which may be due in part, to manual operation of the system 400.The PCM quantification technique was shown to be generally lessreliable, as it is not only indirect, but also introduces multiple humanerrors (e.g., during sample preparation and dilution, plating on agarplates, etc.). Therefore, standard PCM quantification is subjected to alarge statistical error that often goes beyond the +/−0.5 log range.

FIG. 15 illustrates a process for detecting and quantifying bacteria ina sample in accordance with some embodiments. In act 1510, an influentsample is connected to an input pump of the microfluidic system. Theinfluent sample may include a fluorescent dye configured to labelbacteria in the sample to facilitate on-chip quantification.Alternatively, a fluorescent dye may be provided to the microfluidicsystem to label bacteria after they have been captured. In someembodiments, a fluorescent dye is not used. Process 1500 then proceedsto act 1512, where a desired flow rate and electric field parameters areselected. In some embodiments, the flow rate may be selected to achievean expected capture efficiency of bacteria by the electrode. Forinstance, in some embodiments, a flow rate between 10-960 ul/min may beselected to achieve a capture efficiency of at least 99%. In someembodiments, a flow rate between 480-960 ul/min may be selected toachieve a capture efficiency of at least 99%. In some embodiments, aflow rate between 720-960 ul/min may be selected to achieve a captureefficiency of at least 99%. In some embodiments, a flow rate between840-960 ul/min may be selected to achieve a capture efficiency of atleast 99%. In some embodiments, a flow rate may be selected to achieve acapture efficiency of at least 99.6%. In some embodiments, a flow ratebetween 240-480 ul/min may be selected to achieve a capture efficiencyof at least 99.9%. In some embodiments, a flow rate of 240 ul/min may beselected to achieve a capture efficiency of at least 99.99%.

The electric field parameters may be tuned to attract multiple types ofbacteria to the electrode(s) within the microfluidic system or may betuned to selectively capture one or more types of bacterial specieswhile repelling one or more other types of bacterial species. In someembodiments, setting electric field parameters comprises setting anamplitude and/or frequency of a voltage provided to activate the one ormore electrodes within the microfluidic system. In some embodiments, thefrequency of an AC voltage provided to the electrode(s) is within arange between 900 Hz-2 MHz. In some embodiments, the frequency of the ACvoltage is 1 MHz.

Process 1500 then proceeds to act 1514, where the electric field isturned on in accordance with the selected parameters and the influentsample is pumped at the selected flow rate through one or more channelsin the microfluidic device associated with the one or more electrodes.As the sample traverses the portion of the channel(s) proximate to theone or more electrodes, bacteria are captured from the sample on thesurface of the electrode(s) due to a positive DEP force acting on thebacteria in the sample.

Process 1500 then proceeds to act 1516, where a number of bacteriacaptured on the electrode(s) are quantified, for example, by analyzingone or more images captured by an optical system while the bacteria arecaptured by the electrode(s) (e.g., using direct on-chip quantification,as described herein).

Recent discoveries have shown relationships between the human microbiomeand human health. There is a great promise of creating therapeutics thatchange the human microbiome to cure diseases such as obesity, diabetes,autism, bipolar disorder and Alzheimer's disease.

Research and development of new therapeutics requires sequencing humansamples such as fecal samples, skin swabs, vagina swabs, nasal swabs,samples from intestines and the gastrointestinal (GI) tract, mouth andgums swabs.

Most microbiome samples from a human body contain both non-bacterialcells and bacterial species that are present in high abundance. Themajority of DNA extracted from human microbiome samples is from thenon-bacterial cells and bacterial species that are present in highabundance, which makes it challenging to detect the presence ofbacterial or viral species present in low abundance. Although humanmicrobiome samples are used as an example of the type of sample that maybe processed in accordance with some embodiments, it should beappreciated that microbiome samples may alternatively be processed fromplants, soil, water or animals.

Some embodiments are directed to methods and apparatus for detectingbacteria at low concentrations in a complex sample by enriching thebacteria following capture. For instance, some embodiments relate to amethod for enriching for bacterial species from complex samples, such asfecal samples, where the bacterial species of interest are below 0.1% ofthe total bacterial or cellular concentration, which is generallyconsidered the reliable limit of bacterial detection with sequencing. Inparticular, some embodiments relate to a method that uses an electricfield in a microfluidic chip to remove cellular noise from samples andselectively enrich a bacterial species of interest. An application ofsuch techniques is to selectively capture bacterial species in fecalsamples or other complex samples while removing non-bacterial cells andnon-target bacteria (e.g., gram-negative bacteria) from the samples.Such an improved process for detecting low concentrations of bacteriamay provide an automated process for specific enrichment of a bacterialspecies from a fecal sample or another complex sample in minutes tohours, instead of days as is typically required using standardtechniques for detecting bacteria in samples (e.g., PCM).

The inventors have recognized some limitations of conventional moleculartechniques include the inability of such techniques to detect andquantify DNA from a species that is present in a complex sample in lowabundance. Such detection becomes more challenging with an increasingconcentration of foreign DNA in a sample. Increasing the sample volumefurther limits detection capabilities of molecular methods by dilutingthe concentration of the species of interest. Precise detection of a lowconcentration of DNA in a sample has many applications including, butnot limited to liquid biopsy, microbiome therapeutics, and diseasediagnostics.

Tumors release circulating tumor cells into the body. The circulatingtumor cells and DNA coming from the cells are present in low abundance.Sensitive detection of DNA from circulating tumor cells can help detectearly stage tumors, which can lead to diagnosing cancer at an earlystage and potentially improve patient outcomes.

Detecting DNA from circulating tumor cells in blood is challenging,because there are billions of red blood cells and other DNA in everymilliliter of blood, which causes high background noise for cell and DNAdetection. In some embodiments, DNA present in low abundance in a bloodsample is detected by performing a liquid biopsy on the blood samplefollowing enhancement of the DNA of interest using one or more of thetechniques described herein.

Similar to liquid biopsy or human microbiome samples, disease agents atan early stage of a disease are usually present in low abundance. DNAfrom a disease-causing agent may be hard to detect due to the presenceof DNA noise arising from cells and other microbes. In some embodiments,DNA from a disease-causing agent is detected in low abundance in aliquid sample (e.g., blood, urine, saliva) following enhancement of theDNA using one or more of the techniques described herein.

Although the example microorganism described herein is bacteria, itshould be appreciated that other microorganisms including, but notlimited to, yeast, mold and viruses may also be detected in lowabundance using one or more of the techniques for enrichment describedherein.

As discussed in more detail below, enrichment of a target bacterialspecies is achieved by capturing bacteria on one or more electrodes ofthe microfluidic chip by applying an electric field in a preselectedfrequency range, washing away debris and non-target bacteria, andreleasing captured components from the electrodes of the microfluidicchip.

FIG. 16 schematically illustrates a process for enriching bacteria inaccordance with some embodiments. An influent sample 1610 may containmultiple types of bacteria and non-bacterial components in differentconcentrations. Some bacteria may be at such low concentrations thatthey may not be detectable when cultured on a petri dish 1612, forexample, using PCM. Influent sample 1610 is provided as input to amicrofluidic channel of microfluidic device 1614 configured to capturebacteria on one or more electrodes using dielectrophoresis as the sample1610 flows from an inlet of the microfluidic channel to an outlet of themicrofluidic channel. Components of sample 1610 that are not captured bythe electrode(s) in the microfluidic devices exit the outlet of themicrofluidic channel and are collected in effluent sample container 1620for further analysis, if desired. Following capture, the components(e.g., cells) are released from the electrodes and may be output from anoutlet of the microfluidic channel into effluent sample container 1622for further analysis, if desired.

Release of the captured components from the electrodes of themicrofluidic chip may be accomplished in some embodiments using one or acombination of following techniques:

-   -   Captured target bacterial species are released from the        electrodes to an outlet of a microfluidic channel by turning off        the electric field. Turning off the electric field causes the        dielectrophoresis force that captures bacteria to disappear.        Fluid flow through the microfluidic channel washes off bacteria        to an outlet of the microfluidic channel. At the outlet of the        microfluidic channel bacteria are collected for DNA extraction        or further processing for enrichment with bacterial culture or a        growth-based technique.    -   Captured target bacterial species are released from the        electrodes to an outlet of a microfluidic channel by applying a        high frequency electric field to induce negative        dielectrophoresis that repels captured cells including the        target bacterial species from the electrodes. Fluid flow through        the microfluidic channel washes off bacteria to an outlet of the        microfluidic channel. At the outlet of a microfluidic channel        bacteria are collected for DNA extraction or further processing        for enrichment with bacterial culture or a growth-based        technique.    -   Captured target bacterial species are released from the        electrodes to an outlet of a microfluidic channel by flushing        the microchannel of the microfluidic chip with a fluid at a high        pressure. The fluid at a high pressure removes target bacterial        species and DNA adherent to the surface of the electrodes to an        outlet of the microfluidic channel. At the outlet of a        microfluidic channel bacteria are collected for DNA extraction        or further processing for enrichment with bacterial culture or a        growth-based technique.

FIGS. 17A and 17B schematically illustrate two configurations of asystem for, among other things, detecting, quantifying, sorting, andenriching bacteria from microbiome samples in accordance with someembodiments. Influent sample 1701 contains a microbiome sample (e.g., afecal sample, a skin swab sample, etc.). In some embodiments, a fecalsample is prepared for processing using the following process. The fecalsample is diluted 1:10 and emulsified mechanically preserving cellintegrity. The emulsified sample is then filtered through a series ofcell strainers including 100 um, 70 um, 40 um, 20 um and 10 um poresizes. Several volumes of 0.001×PBS are passed through the cellstrainers after the sample to wash the strainers of the sample and bringthe final volume to a 1:100 dilution. Filtration is helpful to preventparticulates present in the fecal sample from clogging the microfluidicdevice. In some embodiments, a skin swab sample is prepared forprocessing using the systems shown in FIGS. 17A and 17B. One or moresamples are collected from a donor using one or more skin swabs placedinto a sterile buffer solution (e.g., 100 mL of 0.001×PBS). Between eachswab collection, a container with the buffer solution may be agitated toensure that the majority of the sample is transferred to the PBS media.The sample for processing may be aspirated (e.g., using a 27-gaugeneedle) from PBS media to high-pressure homogenize the sample.Aspiration in this manner breaks up clumps of skin cells in the samplewithout disturbing the native microbiota that may be adhered to thecells.

In the configuration shown in FIG. 17A, influent sample 1701 isconnected to pump 1702, which is configured to cause the sample 1701 toflow through the inlet 1703 coupled to a microfluidic channel of themicrofluidic device 1704. Arrow 1705 shows the direction of fluid flowthrough the microfluidic channel. The system also includes signalgenerator 1708 configured to generate a fixed signal or a variablefrequency and amplitude signal having a voltage amplitude between 5V and100V and frequency between 100 Hz and 400 MHz. As shown, signalgenerator 1708 is configured to generate electric signal 1706 applied toalternating (e.g., odd-numbered) rings of an electrode disposed withinthe microfluidic channel and electric signal 1707 applied to the otheralternating (e.g., even-numbered) rings of the electrode and having anopposite polarity to electric signal 1706 or ground (e.g., 0V).Activation of the rings of the electrode create an electric field havinga gradient 1709 pointing towards the electrode or electrode edge. As thesample 1701 flows through the microfluidic channel, bacteria of interestare captured onto the surface of the electrode by the electric field,whereas other components in the sample 1701 are not captured by theelectric field and proceed to flow through output 1710 in themicrofluidic channel which is coupled to effluent sample container 1711.

FIG. 17B shows a configuration similar to that described for FIG. 17A,but with pump 1702 located outside of the flow path, such that thesample 1701 does not pass through the pump during processing. In theconfiguration of FIG. 17B the pump creates sub-pressure in the systemthat causes the fluid flow through the microfluidic device asillustrated by arrow 1605.

In accordance with some embodiments, methods and apparatus for enrichingorganisms (e.g., bacteria) in a sample are provided, as shown in FIGS.18A-D. Sample 1804 to be processed may include target bacterial species1801, other bacterial species 1802 (e.g., Bacteroidetes for a fecalsample) and non-bacterial components 1803 (e.g., cells of human, plantor animal origin for a fecal sample; skin cells for a skin swab sample)as shown in FIG. 18A.

FIG. 18B shows a process by which bacteria in the influent sample 1804are separated from non-bacterial microscopic components. The influentsample 1804 is provided to inlet 1805 of a microfluidic channel inmicrofluidic device 1806. As the sample 1804 flows through themicrofluidic channel in the direction shown by arrow 1810, targetbacterial species 1801 and other bacterial species 1802 are attracted toelectrode 1809 due to a positive dielectrophoretic force exerted on thebacterial species as shown by the bacteria moving towards the electrode1809 (see e.g., target bacterial species 1807 and other bacterialspecies 1808). In some embodiments, the amplitude and frequency of an ACvoltage applied to the electrode 1809 to generate an applied electricfield may be selected to attract and capture a broad range of bacteria(e.g., bacteria including, but not exclusive to, the target bacterialspecies 1801) on the electrode. For instance, the frequency of theapplied AC voltage may be in the range 100 kHz-5 MHz and the amplitudeof the applied AC voltage may be in the range 5V-100V peak-to-peak.Identifier 1811 in FIG. 18B schematically shows a trajectory ofbacterial motion towards the electrode 1709 due to the applied electricfield and identifier 1812 shows that non-bacterial components in thesample 1804 are not captured by the electric field, but instead flowtoward outlet 1813 of the microfluidic channel to be collected ineffluent sample container 1814. For instance, effluent sample container1814 collects microscopic components and fluid that passes through themicrofluidic device without being captured by the electrode 1809 whilethe electric field is turned on.

FIG. 18C shows a process for releasing the captured bacteria from theelectrodes and collecting the released bacteria into effluent samplecontainer 1818. One or more characteristics (e.g., amplitude, frequency)of the applied electric field 1815 may be changed to release thecaptured bacteria. For instance, the applied electric field 1815 may bealtered by deactivating the electrodes by turning off the electric fieldor by applying an electric field with different characteristics (e.g., ahigh frequency electric field) that reduces the positivedielectrophoresis force or induces a negative dielectrophoresis force onthe captured bacteria to repel the bacteria from the electrode surface.In some embodiments, the sample matrix may be exchanged to a controlledmatrix (e.g., a buffer solution 0.001×PBS) prior to releasing thecaptured bacteria. Indicators 1816 and 1817 show the captured targetbacterial species and the captured other bacterial species,respectively, moving away from the electrode in response to changes inthe electric field. The fluid flow (e.g., including the introducedbuffer solution) pushes the released bacteria toward effluent samplecontainer 1818, which may be a different effluent sample container thanthe one used to collect waste during capture of the bacteria.

FIG. 18D shows a process for mechanically releasing captured components(e.g., bacterial cells) from the electrodes. A high-pressure washsolution 1820 is flushed through the microfluidic channel of themicrofluidic device to mechanically release cells adhering to theelectrodes (e.g., in the absence of the applied electric field. Thereleased components are collected in the effluent wash sample container1819.

In accordance with some embodiments, methods and apparatus for enrichingorganisms (e.g., a target bacterial species) in a sample are provided,as shown in FIGS. 19A-D. Sample 1904 to be processed may include targetbacterial species 1901, other bacterial species 1902 (e.g.,Bacteroidetes for a fecal sample) and non-bacterial components 1903(e.g., cells of human, plant or animal origin for a fecal sample; skincells for a skin swab sample) as shown in FIG. 19A.

FIG. 19B shows a process by which target bacterial species 1901 in theinfluent sample 1904 are selectively separated from other bacterialspecies 1902 and non-bacterial components 1903 in the sample. Theinfluent sample 1904 is provided to inlet 1905 of a microfluidic channelin microfluidic device 1906. As the sample 1904 flows through themicrofluidic channel in the direction shown by arrow 1910, targetbacterial species 1901 is attracted to electrode 1909 due to a positivedielectrophoretic force exerted on the target bacterial species as shownby the target bacterial species moving towards the electrode 1909 (seee.g., target bacterial species 1907). In some embodiments, the amplitudeand frequency of an AC voltage applied to the electrode 1909 to generatean applied electric field may be selected to attract and selectivelycapture target bacterial species 1901 on the electrode without capturingother components. For instance, the frequency of the applied AC voltagemay be in the range 2 MHz-5 MHz and the amplitude of the applied ACvoltage may be in the range 5V-100V peak-to-peak. Identifier 1911 inFIG. 19B schematically shows a trajectory of the motion of the targetbacterial species 1901 towards the electrode 1909 due to the appliedelectric field and identifier 1912 shows that other bacterial components1902 and non-bacterial components 1903 in the sample 1904 are notcaptured by the electric field, but instead flow toward outlet 1913 ofthe microfluidic channel to be collected in effluent sample container1914. For instance, effluent sample container 1914 collects microscopiccomponents and fluid that passes through the microfluidic device withoutbeing captured by the electrode 1909 while the electric field is turnedon.

FIG. 19C shows a process for releasing the captured target bacterialspecies from the electrodes and collecting the released bacteria intoeffluent sample container 1917. One or more characteristics (e.g.,amplitude, frequency) of the applied electric field 1915 may be changedto release the captured target bacterial species. For instance, theapplied electric field 1915 may be altered by deactivating theelectrodes by turning off the electric field or by applying an electricfield with different characteristics (e.g., a high frequency electricfield) that reduces the positive dielectrophoresis force or induces anegative dielectrophoresis force on the captured bacteria to repel thebacteria from the electrode surface. In some embodiments, the samplematrix may be exchanged to a controlled matrix (e.g., a buffer solution0.001×PBS) prior to releasing the captured bacteria. Indicator 1916indicates that the captured target bacterial species moves away from theelectrode in response to changes in the electric field. The fluid flow(e.g., including the introduced buffer solution) pushes the releasedbacteria toward effluent sample container 1917, which may be a differenteffluent sample container than the one used to collect waste duringcapture of the bacteria.

FIG. 19D shows a process for mechanically releasing captured anyremaining target bacterial species 1901 from the electrodes. Ahigh-pressure wash solution 1919 is flushed through the microfluidicchannel of the microfluidic device to mechanically release cellsadhering to the electrodes (e.g., in the absence of the applied electricfield). The released components are collected in the effluent washsample container 1918.

In accordance with some embodiments, methods and apparatus for enrichingmultiple organisms (e.g., a first target bacterial species and a secondtarget bacterial species) in a sample are provided, as shown in FIGS.20A-E. For instance, the processes described in FIGS. 18A-D and 19A-Dmay be combined. In some embodiments, the steps shown in FIGS. 19A-D maybe repeated multiple times with multiple runs being applied sequentiallyon separate microfluidic devices or being applied on the samemicrofluidic device with an electrode geometry that allows forapplication of a sequence of electric field signals with controlledamplitude and/or frequency to separate multiple target bacterialspecies. Selectivity may be achieved by selectively releasing speciesthat are not target species of interest.

Sample 2004 to be processed may include first target bacterial species2001 (e.g., E. coli in a fecal sample), second target bacterial species2002 (e.g., Bacteroidetes for a fecal sample) and non-bacterialcomponents 2003 (e.g., cells of human, plant or animal origin for afecal sample; skin cells for a skin swab sample) as shown in FIG. 20A.

FIG. 20B shows a process by which first target bacterial species 2001 inthe influent sample 2004 is selectively separated from second targetbacterial species 2002 and non-bacterial components 2003 in the sample.The influent sample 2004 is provided to inlet 2005 of a microfluidicchannel in microfluidic device 2006. As the sample 2004 flows throughthe microfluidic channel in the direction shown by arrow 2010, bothfirst target bacterial species 2001 and second target bacterial species2002 are attracted to electrode 2009 due to a positive dielectrophoreticforce exerted on the first and second target bacterial species as shownby the first and second target bacterial species moving towards theelectrode 2009 (see e.g., first target bacterial species 2007 and secondbacterial species 2008). In some embodiments, the amplitude andfrequency of an AC voltage applied to the electrode 2009 to generate anapplied electric field may be selected to attract both first targetbacterial species 2001 and second target bacterial species 2002 on theelectrode without capturing other components. For instance, thefrequency of the applied AC voltage may be in the range 100 kHz-5 MHzand the amplitude of the applied AC voltage may be in the range 5V-100Vpeak-to-peak. Identifier 2011 in FIG. 20B schematically shows atrajectory of the motion of the target bacterial species towards theelectrode 2009 due to the applied electric field and identifier 2012shows that non-bacterial components 2003 in the sample 2004 are notcaptured by the electric field, but instead flow toward outlet 2013 ofthe microfluidic channel to be collected in effluent sample container2014. For instance, effluent sample container 2014 collects microscopiccomponents and fluid that passes through the microfluidic device withoutbeing captured by the electrode 2009 while the electric field is turnedon.

FIG. 20C shows a process for releasing one of the two captured targetbacterial species from the electrodes and collecting the releasedbacteria into effluent sample container 2018. One or morecharacteristics (e.g., amplitude, frequency) of the applied electricfield 2015 may be changed to selectively release one of the two capturedtarget bacterial species from the electrode. For instance, the appliedelectric field 2015 may be altered by applying an electric field withdifferent characteristics (e.g., an electric field with a differentfrequency) that reduces the positive dielectrophoresis force or inducesa negative dielectrophoresis force on either the first or the secondcaptured bacteria to repel the bacteria from the electrode surface. Insome embodiments, the sample matrix may be exchanged to a controlledmatrix (e.g., a buffer solution 0.001×PBS) prior to releasing thecaptured bacteria. Indicator 2016 indicates that the captured secondtarget bacterial species moves away from the electrode in response tochanges in the electric field. The fluid flow (e.g., including theintroduced buffer solution) pushes the released bacteria toward effluentsample container 2018, which is a different effluent sample containerthan the one used to collect waste during capture of the bacteria.Indicator 2016 indicates that the first target bacterial species remainscaptured by the electrode.

FIG. 20D shows a process for releasing the captured first targetbacterial species from the electrodes and collecting the releasedbacteria into effluent sample container 2021. One or morecharacteristics (e.g., amplitude, frequency) of the applied electricfield 2019 may be changed to release the captured first target bacterialspecies from the electrode. For instance, the applied electric field2019 may be altered by turning the electric field off or by applying anelectric field with different characteristics (e.g., an electric fieldwith a different frequency) that reduces the positive dielectrophoresisforce or induces a negative dielectrophoresis force on the capturedfirst target bacterial species to repel the bacteria from the electrodesurface. In some embodiments, the sample matrix may be exchanged to acontrolled matrix (e.g., a buffer solution 0.001×PBS) prior to releasingthe captured bacteria. Indicator 2010 indicates that the captured firsttarget bacterial species moves away from the electrode in response tochanges in the electric field. The fluid flow (e.g., including theintroduced buffer solution) pushes the released bacteria toward effluentsample container 2021, which is a different effluent sample containerthan the one used to collect the second target bacterial species orwaste collected during capture of the bacteria.

FIG. 20E illustrates a process for mechanically releasing captured anyremaining bacterial species from the electrodes. A high-pressure washsolution 2023 is flushed through the microfluidic channel of themicrofluidic device to mechanically release cells adhering to theelectrodes (e.g., in the absence of the applied electric field). Thereleased components are collected in the effluent wash sample container2022.

FIG. 21A illustrates results from an experiment in which the limit ofdetection for a target bacterial species C was enhanced. An aliquot ofan influent sample was sequenced and all species having a relativeabundance lower than 0.001 were considered not detectable. The resultsshow no detectable presence of species C in the influent sample. Theinfluent sample was then processed by the microfluidic device with anelectric field having characteristics of frequency between 100 kHz-25MHz at an amplitude between 5-50V and the effluent sample containing thereleased bacteria captured on the electrode was sequenced. The resultsshow the relative abundance of species C being over 0.005, which is overfive times above the detectable limit of 0.001. This result was repeatedfor four different bacterial species.

FIG. 21B illustrates results from another experiment in which theinfluent sample containing a mixture of bacterial species A andbacterial species B was sequenced. The results show the relativeabundance of the two bacterial species is about equal in the influentsample (i.e., the sample prior to processing by the microfluidicdevice). The influent sample was then processed using the microfluidicdevice with an electric field having an operating frequency whichselectively captured species A but not species B. The released effluentsample was sequenced and the results show the relative abundance ofspecies A was close to 25 times higher than that of species B,demonstrating that some embodiments are capable of enriching, on aspecies level, about 25 times relative to the background in the sample.

Microbiome therapeutics often consist of lyophilized consortia ofmultiple bacterial strains. To preserve therapeutic properties, it isimportant that manufacturers ensure reproducibility of the manufacturingprocess. The lyophilized bacterial strains need to yield live bacteriaafter the therapeutic is administered to a patient. This requirescontrol and reproducibility of a manufacturing process. It also requiresensuring that each batch of microbiome drugs has the same ratio andviability of the strains within a consortium.

Controlling the manufacturing processes using current methods ischallenging. Bacterial culture requires at least two days for most ofthe bacterial strains used in such processes, which results in processdelays due to the time it takes to receive feedback on the process.Additionally, it can be challenging to ensure reproducibility ofmanufacturing processes where bacterial levels within a bioreactor canvary by about 100 from batch to batch.

Stain based methods and flow cytometry suffer from high error rates.Additionally, molecular methods do not differentiate between live anddead bacteria. Some embodiments relate to an accurate and real timein-process test that enables more efficient in-process control andensure reproducibility of manufacturing within an acceptable +/−0.5 logvariability range.

Manufacturing microbiome therapeutics requires controlling thecomposition of the final product. The final product release testrequires quantifying the number of bacteria in a pill afterlyophilization by strain and also the number of viable bacteria.Bacterial strains included in a microbiome therapeutic are often closelyrelated and cannot be grown on selective media. This can make itchallenging to use bacterial culture as a method for the final productrelease test. Some embodiments address at least some of these challengesby (1) enabling differentiation and/or quantification of live and deadbacteria in a sample and (2) enabling differentiation and/orquantification of bacteria from a complex mixture.

Some embodiments relate to methods and apparatus for sorting multiplebacterial species in a complex sample using dielectrophoresis. Theinventors have recognized and appreciated that different bacterialspecies are attracted (due to positive dielectrophoresis) or repulsed(due to negative dielectrophoresis) from the surface of an electrodebased on the frequency of an AC voltage applied to the electrode. Withincertain frequency ranges (e.g., 900 Hz-2 MHz) multiple species ofbacteria respond similarly, being attracted to the electrode due topositive dielectrophoresis. Within other higher frequency ranges somebacterial species experience positive dielectrophoresis whereas otherbacterial species experience negative dielectrophoresis. The inventorshave recognized that this differential response, especially at higherfrequencies, may be used to sort bacteria by selecting stimulationfrequencies in which one bacterial species is attracted to the electrodeand one or more other bacterial species are repulsed.

FIG. 22A illustrates a schematic of a microfluidic system in which acomplex mixture of four bacterial species is sorted in accordance withsome embodiments. The microfluidic device shown in FIG. 22A includesmultiple electrodes, each of which can be tuned to provide an electricfield that selectively captures one of the bacterial species in thecomplex mixture on the electrode while other components of the mixturepass to the next electrode in the sequential chain. By using threeelectrodes, each tuned with different electronic conditions, the fourbacterial species in the complex mixture can be separated.

FIG. 22B illustrates a top view of an example microfluidic deviceincluding four electrode systems that may be used to perform bacteriasorting in accordance with some embodiments. The microfluidic systemincludes a microfluidic channel having an inlet 2201, an outlet 2214 anda wall 2215. The microfluidic channel may have any suitable dimensions.Arranged between inlet 2201 and outlet 2214 along the fluid flow pathare first electrode system 2202, second electrode system 2203, thirdelectrode system 2204 and fourth electrode system 2205. First electrodesystem 2202 is coupled to electric contact 2206 configured to supply anAC voltage (voltage: +V1, frequency: f1) and electric contact 2207configured to supply an AC voltage (voltage: −V1 (or ground), frequency:f1). Second electrode system 2203 is coupled to electric contact 2208configured to supply an AC voltage (voltage: +V2, frequency: f2) andelectric contact 2209 configured to supply an AC voltage (voltage: −V2(or ground), frequency: f2). Third electrode system 2204 is coupled toelectric contact 2210 configured to supply an AC voltage (voltage: +V3,frequency: f3) and electric contact 2211 configured to supply an ACvoltage (voltage: −V3 (or ground), frequency: f3). Fourth electrodesystem 2204 is coupled to electric contact 2212 configured to supply anAC voltage (voltage: +V4, frequency: f4) and electric contact 2213configured to supply an AC voltage (voltage: −V4 (or ground), frequency:f4).

In an experiment, the results of which are shown in FIG. 22C, it wasdemonstrated that a microfluidic device designed in accordance with thetechniques described herein can discriminate between four closelyrelated bacterial species—B. subtilis, B. cereus, B. coagulans and B.megaterium by changing the frequency characteristic of the appliedelectric field when the amplitude of the applied AC voltage is heldconstant. Each bacterial species was suspended separately in testedbuffer (PBS 1:1000 in deionized water) and stained with SybrGreen I dye.After 30 minutes of incubation in darkness 2 μL of the sample was loadedonto a microfluidic chip designed in accordance with the techniquesdescribed herein and was visualized using the static system 500described in connection with FIG. 5. The frequency of the appliedelectric field was swept from a low frequency to a high frequency, andwhen certain frequencies were reached particular bacterial speciesresponded to the electric field in characteristic manner in whichloosely floating bacteria (when electric field is off) were forced fromany area of chip to the edges of electrodes (when electric field is on),thereby exhibiting positive dielectrophoresis. When the electric fieldwas turned off the bacteria were released from the edges of theelectrode. In particular, the plot in FIG. 22C shows that the closelyrelated species respond to the electric field in a differential way andthese electronic conditions are characteristic for specific species.

In particular, FIG. 22C illustrates a plot of four different bacterialspecies and their respective cross-over frequency responses for aconstant amplitude AC voltage. For each of the bacterial species, itscross-over frequency is the frequency at which the bacterial speciesswitches from exhibiting positive dielectrophoresis below the cross-overfrequency and negative dielectrophoresis above the cross-over frequency.For instance, as shown in FIG. 22C, the bacterial species B. megateriumhas a lower cross-over frequency than B. subtilis, which has a lowercross-over frequency than B. cerus, which has a lower cross-overfrequency than B. coagulans. By setting the frequency of the appliedelectric field to a frequency that falls between two cross-overfrequencies of different bacterial species, one species will beattracted to the electrode via positive dielectrophoresis while theother species will be repelled via negative dielectrophoresis, therebyenabling separation of the two species.

In accordance with some embodiments, methods and apparatus forseparating organisms (e.g., bacteria) in a sample are provided, as shownin FIGS. 23A-C. An influent sample 2305 to be processed may be, forexample, a microbiome sample containing a bacterial consortium withmultiple bacterial strains. For instance, sample 2305 may include afirst target bacterial species 2301 (e.g., E. coli), a second targetbacterial species 2302 (e.g., B. cerus) and a third target bacterialspecies 2303 (e.g., B. megaterium) as shown in FIG. 23A.

FIG. 23B shows a process by which bacteria in the sample 2305 areseparated and captured on different electrode systems. The sample 2305is provided to inlet 2304 of a microfluidic channel in microfluidicdevice 2306. As the sample 2305 flows through the microfluidic channelin the direction shown by arrow 2313, the first target bacterial speciesis captured using positive dielectrophoresis by a first electrode system2307 being driven by an AC voltage having amplitude and frequencycharacteristics (V1;f1). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in first electrode system 2307 as(+V1, f1; −V1, f1) or (+V1, f1; 0V) to generate an electric field 2310which acts to capture the first bacterial species on the surface of theelectrodes in first electrode system 2307, as illustrated by indicator2314). As illustrated by indicators 2315 and 2316, the second targetbacterial species and the third target bacterial species, respectively,are not captured by the first electrode system 2307, but continueflowing through the microfluidic device 2306.

As the sample 2305 flows through the microfluidic channel in thedirection shown by arrow 2313, the second target bacterial species iscaptured using positive dielectrophoresis by a second electrode system2308 being driven by an AC voltage having amplitude and frequencycharacteristics (V2;f2). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in second electrode system 2308 as(+V2, f2; −V2, f2) or (+V2, f2; 0V) to generate an electric field 2311which acts to capture the second bacterial species on the surface of theelectrodes in second electrode system 2308, as illustrated by indicator2317). As illustrated by indicator 2318, the third target bacterialspecies is not captured by the second electrode system 2308, butcontinues flowing through the microfluidic device 2306.

As the sample 2305 flows through the microfluidic channel in thedirection shown by arrow 2313, the third target bacterial species iscaptured using positive dielectrophoresis by a third electrode system2309 being driven by an AC voltage having amplitude and frequencycharacteristics (V3;f3). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in second electrode system 2309 as(+V3, f3; −V3, f3) or (+V3, f3; 0V) to generate an electric field 2312which acts to capture the third bacterial species on the surface of theelectrodes in third electrode system 2309, as illustrated by indicator2319).

Any remaining components not captured by one of the electrode systemsflow through outlet 2320 into effluent sample container 2321. Forinstance, the effluent sample may contain microscopic components andfluid that passes the microfluidic device while the electric fields areturned on. It should be appreciated that although a three electrodesystem has been illustrated, any number of electrode systems (includingfewer or more electrode systems) to separate any number of organisms inthe sample, and embodiments are not limited in this respect.

FIG. 23C shows a process for quantifying captured bacteria, selectivelyreleasing the captured bacteria from the electrodes, and collecting thereleased bacteria into effluent sample container 2332. Influent samplecontainer 2322 containing a fluid may be connected to the inlet 2304 ofthe microfluidic device. For instance, the fluid in influent samplecontainer 2322 may be used to flush the microfluidic device with acontrolled matrix such as a buffer solution. Additionally oralternatively, the microfluidic device may be flushed with a fluorescentstain or a label and incubated for a period of time.

As shown, the system includes optical system 2329 configured to capturean image of bacteria captured on an electrode. For instance, opticalsystem 2329 may include an optical sensor with a fluorescent lightdetector, such as a fluorescent microscope or light emitting diode (LED)light source 2330 with an objective and a detector. Light source 2330may be configured to excite a fluorophore in the labeled bacteria. Itshould be appreciated that not all embodiments use fluorescent labelingof bacteria or other captured organism, as some embodiments areconfigured to generate bright field images.

Optical system 2329 may be configured to sequentially image the firstelectrode system, the second electrode system, and the third electrodesystem to record, for each electrode system, a fluorescent signal and/oran image corresponding to the electrode system while the fluorescentlystained target bacterial species remain captured by the electrodes inthe electrode system (as shown by indicators 2326, 2327 and 2328).Computer 2331 or another image processor is configured to process imagescaptured by optical system 2329 to recognize bacteria in the capturedimages and to quantify a number of bacteria in the images.

One or more characteristics (e.g., amplitude, frequency) of the appliedelectric fields generated by the electrode systems 2323, 2324 and 2325may be changed to release the captured bacteria from the electrodes. Forinstance, one or more of the applied electric fields may be altered bydeactivating the electrodes of a corresponding electrode system byturning off the electric field or by applying an electric field withdifferent characteristics (e.g., a high frequency electric field) thatreduces the positive dielectrophoresis force or induces a negativedielectrophoresis force on the captured bacteria to repel the bacteriafrom the electrode surface of the electrode system. In some embodiments,the sample matrix may be exchanged to a controlled matrix (e.g., abuffer solution 0.001×PBS) prior to releasing the captured bacteria.Fluid flow in the microfluidic device may push the released bacteriatoward effluent sample container 2332, which may be a different effluentsample container than the one used to collect waste during capture ofthe bacteria.

In accordance with some embodiments, methods and apparatus forseparating organisms (e.g., bacteria) in a sample are provided, as shownin FIGS. 24A-C. An influent sample 2405 to be processed may be, forexample, a microbiome sample containing a bacterial consortium withmultiple bacterial strains and other particles. For instance, sample2405 may include a first target bacterial species 2401 (e.g., E. coli),a second target bacterial species 2402 (e.g., B. cerus), a third targetbacterial species 2403 (e.g., B. megaterium), and non-bacterialcomponents or bacteria that are not of interest in a sample (e.g., cellsof human, plant or animal origin in a fecal sample, skin cells in a skinswab sample, or components of growth media from a bioreactor), as shownin FIG. 24A.

FIG. 24B shows a process by which bacteria in the sample 2405 areseparated from the non-bacterial components and are captured ondifferent electrode systems. The sample 2405 is provided to an inlet ofa microfluidic channel in microfluidic device 2406. As the sample 2405flows through the microfluidic channel in the direction shown by arrow2413, the first target bacterial species is captured using positivedielectrophoresis by a first electrode system 2407 being driven by an ACvoltage having amplitude and frequency characteristics (V1;f1). Inparticular, the AC voltage signal is applied to electrodes of oppositepolarity in first electrode system 2407 as (+V1, f1; −V1, f1) or (+V1,f1; 0V) to generate an electric field 2410 which acts to capture thefirst bacterial species on the surface of the electrodes in firstelectrode system 2407, as illustrated by indicator 2414). As illustratedby indicators 2415, 2416, and 2420, the second target bacterial species,the third target bacterial species, and the other components,respectively, are not captured by the first electrode system 2407, butcontinue flowing through the microfluidic device 2406.

As the sample 2405 flows through the microfluidic channel in thedirection shown by arrow 2413, the second target bacterial species iscaptured using positive dielectrophoresis by a second electrode system2408 being driven by an AC voltage having amplitude and frequencycharacteristics (V2;f2). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in second electrode system 2408 as(+V2, f2; −V2, f2) or (+V2, f2; 0V) to generate an electric field 2411which acts to capture the second bacterial species on the surface of theelectrodes in second electrode system 2408, as illustrated by indicator2417). As illustrated by indicators 2418 and 2421, the third targetbacterial species and the other components, respectively, are notcaptured by the second electrode system 2408, but continues flowingthrough the microfluidic device 2406.

As the sample 2405 flows through the microfluidic channel in thedirection shown by arrow 2413, the third target bacterial species iscaptured using positive dielectrophoresis by a third electrode system2409 being driven by an AC voltage having amplitude and frequencycharacteristics (V3;f3). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in second electrode system 2409 as(+V3, f3; −V3, f3) or (+V3, f3; 0V) to generate an electric field 2412which acts to capture the third bacterial species on the surface of theelectrodes in third electrode system 2409, as illustrated by indicator2419).

Any remaining components not captured by one of the electrode systemsflow through an outlet of the microfluidic device into effluent samplecontainer 2423. For instance, the effluent sample may containmicroscopic components and fluid that passes the microfluidic devicewhile the electric fields are turned on. It should be appreciated thatalthough a three electrode system has been illustrated, any number ofelectrode systems (including fewer or more electrode systems) toseparate any number of organisms in the sample, and embodiments are notlimited in this respect.

FIG. 24C shows a process for quantifying captured bacteria, selectivelyreleasing the captured bacteria from the electrodes, and collecting thereleased bacteria into effluent sample container 2432. Influent samplecontainer 2434 containing a fluid may be connected to the inlet of themicrofluidic device. For instance, the fluid in influent samplecontainer 2434 may be used to flush the microfluidic device with acontrolled matrix such as a buffer solution. Additionally oralternatively, the microfluidic device may be flushed with a fluorescentstain or a label and incubated for a period of time.

As shown, the system includes optical system 2429 configured to capturean image of bacteria captured on an electrode. For instance, opticalsystem 2429 may include an optical sensor with a fluorescent lightdetector, such as a fluorescent microscope or light emitting diode (LED)light source 2430 with an objective and a detector. Light source 2430may be configured to excite a fluorophore in the labeled bacteria. Itshould be appreciated that not all embodiments use fluorescent labelingof bacteria or other captured organism, as some embodiments areconfigured to generate bright field images.

Optical system 2429 may be configured to sequentially image the firstelectrode system, the second electrode system, and the third electrodesystem to record, for each electrode system, a fluorescent signal and/oran image corresponding to the electrode system while the fluorescentlystained target bacterial species remain captured by the electrodes inthe electrode system (as shown by indicators 2426, 2427 and 2428).Computer 2431 or another image processor is configured to process imagescaptured by optical system 2429 to recognize bacteria in the capturedimages and to quantify a number of bacteria in the images.

One or more characteristics (e.g., amplitude, frequency) of the appliedelectric fields generated by the electrode systems 2433, 2424 and 2425may be changed to release the captured bacteria from the electrodes. Forinstance, one or more of the applied electric fields may be altered bydeactivating the electrodes of a corresponding electrode system byturning off the electric field or by applying an electric field withdifferent characteristics (e.g., a high frequency electric field) thatreduces the positive dielectrophoresis force or induces a negativedielectrophoresis force on the captured bacteria to repel the bacteriafrom the electrode surface of the electrode system. In some embodiments,the sample matrix may be exchanged to a controlled matrix (e.g., abuffer solution 0.001×PBS) prior to releasing the captured bacteria.Fluid flow in the microfluidic device may push the released bacteriatoward effluent sample container 2432, which may be a different effluentsample container than the one used to collect waste during capture ofthe bacteria.

In accordance with some embodiments, methods and apparatus forseparating live from dead organisms (e.g., bacteria) in a sample areprovided, as shown in FIGS. 25A-C. An influent sample 2503 to beprocessed may include a first target bacterial species 2501 (e.g., liveE. coli) and a second target bacterial species 2502 (e.g., dead E. coli)as shown in FIG. 25A.

FIG. 25B shows a process by which live and dead bacteria in the sample2503 are separated and captured on different electrode systems. Thesample 2503 is provided to inlet 2504 of a microfluidic channel inmicrofluidic device 2506. As the sample 2503 flows through themicrofluidic channel in the direction shown by arrow 2505, the firsttarget bacterial species is captured using positive dielectrophoresis bya first electrode system 2507 being driven by an AC voltage havingamplitude and frequency characteristics (V1;f1). In particular, the ACvoltage signal is applied to electrodes of opposite polarity in firstelectrode system 2507 as (+V1, f1; −V1, f1) or (+V1, f1; 0V) to generatean electric field 2509 which acts to capture the first target bacterialspecies on the surface of the electrodes in first electrode system 2507,as illustrated by indicator 2511). As illustrated by indicator 2512, thesecond target bacterial species is not captured by the first electrodesystem 2507, but continues flowing through the microfluidic device 2506.

As the sample 2503 flows through the microfluidic channel in thedirection shown by arrow 2505, the second target bacterial species iscaptured using positive dielectrophoresis by a second electrode system2508 being driven by an AC voltage having amplitude and frequencycharacteristics (V2;f2). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in second electrode system 2508 as(+V2, f2; −V2, f2) or (+V2, f2; 0V) to generate an electric field 2510which acts to capture the second bacterial species on the surface of theelectrodes in second electrode system 2508, as illustrated by indicator2513).

Any remaining components not captured by one of the electrode systemsflow through outlet 25140 into effluent sample container 2515. Forinstance, the effluent sample may contain microscopic components andfluid that passes the microfluidic device while the electric fields areturned on.

FIG. 25C shows a process for quantifying captured bacteria, selectivelyreleasing the captured bacteria from the electrodes, and collecting thereleased bacteria into effluent sample container 2524. Influent samplecontainer 2516 containing a fluid may be connected to the inlet of themicrofluidic device. For instance, the fluid in influent samplecontainer 2516 may be used to flush the microfluidic device with acontrolled matrix such as a buffer solution. Additionally oralternatively, the microfluidic device may be flushed with a fluorescentstain or a label and incubated for a period of time.

As shown, the system includes optical system 2521 configured to capturean image of bacteria captured on an electrode. For instance, opticalsystem 2521 may include an optical sensor with a fluorescent lightdetector, such as a fluorescent microscope or light emitting diode (LED)light source 2522 with an objective and a detector. Light source 2522may be configured to excite a fluorophore in the labeled bacteria. Itshould be appreciated that not all embodiments use fluorescent labelingof bacteria or other captured organism, as some embodiments areconfigured to generate bright field images.

Optical system 2521 may be configured to sequentially image the firstelectrode system and the second electrode system to record, for eachelectrode system, a fluorescent signal and/or an image corresponding tothe electrode system while the fluorescently stained target bacterialspecies remains captured by the electrodes in the electrode system (asshown by indicators 2519 and 2520). Computer 2523 or another imageprocessor is configured to process images captured by optical system2521 to recognize bacteria in the captured images and to quantify anumber of bacteria in the images.

One or more characteristics (e.g., amplitude, frequency) of the appliedelectric fields generated by the electrode systems 2517 and 2518 may bechanged to release the captured bacteria from the electrodes. Forinstance, one or more of the applied electric fields may be altered bydeactivating the electrodes of a corresponding electrode system byturning off the electric field or by applying an electric field withdifferent characteristics (e.g., a high frequency electric field) thatreduces the positive dielectrophoresis force or induces a negativedielectrophoresis force on the captured bacteria to repel the bacteriafrom the electrode surface of the electrode system. In some embodiments,the sample matrix may be exchanged to a controlled matrix (e.g., abuffer solution 0.001×PBS) prior to releasing the captured bacteria.Fluid flow in the microfluidic device may push the released bacteriatoward effluent sample container 2524, which may be a different effluentsample container than the one used to collect waste during capture ofthe bacteria.

In accordance with some embodiments, methods and apparatus forseparating live from dead organisms (e.g., bacteria) in a sample areprovided, as shown in FIGS. 26A-C. An influent sample 2603 to beprocessed may include a first target bacterial species 2601 (e.g., liveE. coli) and a second target bacterial species 2602 (e.g., dead E. coli)as shown in FIG. 26A.

FIG. 26B shows a process by which live and dead bacteria in the sample2603 are separated and either the live bacteria or the dead bacteria arecaptured on an electrode system. The sample 2603 is provided to inlet2604 of a microfluidic channel in microfluidic device 2609. The firsttarget bacterial species in the sample 2603 is captured using positivedielectrophoresis by an electrode system 2605 being driven by an ACvoltage having amplitude and frequency characteristics (V1;f1). Inparticular, the AC voltage signal is applied to electrodes of oppositepolarity in electrode system 2605 as (+V1, f1; −V1, f1) or (+V1, f1; 0V)to generate an electric field 2606, which acts to capture the firsttarget bacterial species on the surface of the electrodes in electrodesystem 2605, as illustrated by indicator 2607). As illustrated byindicator 2608, the AC voltage characteristics are selected such thatthe second target bacterial species is not captured by the electrodesystem 2605.

As shown, the system includes optical system 2611 configured to capturean image of bacteria captured on an electrode. For instance, opticalsystem 2611 may include an optical sensor with a fluorescent lightdetector, such as a fluorescent microscope or light emitting diode (LED)light source 2610 with an objective and a detector. Light source 2610may be configured to excite a fluorophore in the labeled bacteria. Itshould be appreciated that not all embodiments use fluorescent labelingof bacteria or other captured organism, as some embodiments areconfigured to generate bright field images.

Optical system 2611 may be configured to image electrode system 2605 torecord a fluorescent signal and/or an image corresponding to theelectrode system while the fluorescently stained target bacterialspecies remains captured by the electrodes in the electrode system (asshown by indicator 2615). Computer 2612 or another image processor isconfigured to process image(s) captured by optical system 2611 torecognize bacteria in the captured image(s) and to quantify a number ofbacteria in the image(s).

FIG. 26C shows a process for capturing both the first target bacterialspecies and the second target bacterial species on the electrode system.Both the first target bacterial species and the second target bacterialspecies are captured using positive dielectrophoresis by an electrodesystem 2613 being driven by an AC voltage having amplitude and frequencycharacteristics (V2;f2). In particular, the AC voltage signal is appliedto electrodes of opposite polarity in electrode system 2613 as (+V2, f2;−V2, f2) or (+V2, f2; 0V) to generate an electric field 2614, which actsto capture both the first target bacterial species and the second targetbacterial species on the surface of the electrodes in electrode system2613, as illustrated by indicator 2615). In some embodiments V2 isbetween 1 V and 100V and f2 is between 100 kHz and 20 MHz.

As shown, the system includes optical system 2616 configured to capturean image of bacteria captured on an electrode. For instance, opticalsystem 2616 may include an optical sensor with a fluorescent lightdetector, such as a fluorescent microscope or light emitting diode (LED)light source 2617 with an objective and a detector. Light source 2617may be configured to excite a fluorophore in the labeled bacteria. Itshould be appreciated that not all embodiments use fluorescent labelingof bacteria or other captured organism, as some embodiments areconfigured to generate bright field images.

Optical system 2616 may be configured to image electrode system 2613 torecord a fluorescent signal and/or an image corresponding to theelectrode system while the fluorescently stained target bacterialspecies remains captured by the electrodes in the electrode system (asshown by indicator 2615). Computer 2618 or another image processor isconfigured to process image(s) captured by optical system 2616 torecognize bacteria in the captured image(s) and to quantify a number ofbacteria in the image(s).

In another experiment, bacteria from two different genera suspended in(PBS 1:1000 in deionized water) and stained with SybrGreen I were shownto respond to the applied electric field differently. E. coli and B.megaterium were suspended in tested buffer, stained with SybrGreen I dyeseparately and after 30 minutes of incubation in darkness were combinedand loaded to the microfluidic chip described previously in system 500of FIG. 5. When certain voltage and frequency for B. megaterium werereached bacteria responded to the electric field in characteristicmanner by being captured on the surface of the electrode, which ischaracteristic of positive dielectrophoresis, while E. coli was notattracted to the electrodes. When electronic conditions werespecifically changed, E. coli also responded by responding to theelectric field and were attracted to the edges of electrodes, therebyexhibiting positive dielectrophoresis.

The same set of experiments was performed for mixtures of E. coli/B.cereus, E. coli/B. coagulans and E. coli/B. subtilis. For eachsuccessive pair of Gram (−) and Gram (+) bacteria, the same behavior wasobserved. Depending on electrical conditions was capture Bacillus spp.or both genera.

In subsequent experiments it was demonstrated that even closely relatedspecies respond to the electric field differently. The B. cereus and B.coagulans were suspended in tested buffer, stained with SybrGreen I dyeseparately and after 30 minutes of incubation in darkness were combinedand loaded to the microfluidic chip and visualized using the staticsystem 500 shown in FIG. 5. When a certain voltage and frequency for B.cereus was reached, bacteria responded to the electric field in acharacteristic manner, while B. coagulans was not attracted to theelectrodes. When electronic conditions were changed, B. coagulans alsoresponded to the electric field and was captured on the edges ofelectrodes.

The same set of experiments was also performed for mixtures of B.megaterium and B. subtilis. As described above at a certain voltage andfrequency B. megaterium was captured while B. subtilis did not respondto the electric field. The change in electrical conditions resulted incapture both B. megaterium and B. subtilis.

FIGS. 27A-27E illustrate images showing selective capture of B.megaterium (shown as long strands) and B. subtilis (shown as dots) inaccordance with the techniques described above. FIG. 27A illustrates animage taken at electric field settings (90 MHz, 20 Vpp) that correspondto positive dielectrophoresis for B. megaterium. B. megaterium bacteriaare captured at the electrode edges, while B. subtilis is not captured,but instead floats above the electrodes. FIG. 27D illustrates a zoomedin version of the electrode in which the electric field was tuned tocapture B. megaterium (which is in focus in the image, but not B.subtilis (which is out of focus).

FIG. 27B illustrates an image taken when the electric field (20 MHz, 20Vpp) was turned on as to achieve positive dielectrophoresis for B.megaterium and B. subtilis. Both species are captured on the electrodeedges. B. megaterium orientation under both electronic conditions inFIG. 27A and FIG. 27B is tangential (parallel) to the electrode edge.FIG. 27E illustrates a zoomed in version of the electrode in which theelectric field is tuned such that both B. megaterium and B. subtilis arecaptured.

FIG. 27C illustrates an image taken when the frequency of the electricfield is adjusted in the low frequency range (1 MHz, 20 Vpp). Only B.subtilis remains captured on the edges of the electrode while B.megaterium switched orientation from tangential (parallel) to orthogonalto the electrode edge.

FIGS. 28A and 28B illustrate images showing selective capture of B.megaterium (shown as long strands) from E. coli (shown as dots). FIG.28A illustrates an image taken when the frequency of the electric fieldis 50 Vpp and 90 MHz tuned to capture only B. megaterium but not E.coli, which is out of focus. FIG. 28B illustrates an image taken whenthe frequency of the electric field is 50 Vpp and 20 MHz tuned tocapture both B. megaterium and E. coli.

Bacteria detected in samples using stain-based methods or molecularmethods are no longer viable. By contrast, the inventors have recognizedthat detection of bacteria using one or more of the techniques describedherein (e.g., using dielectrophoresis capture) yields viable bacteriafollowing their capture (i.e., capture of the bacteria does not kill thebacteria). Accordingly, some embodiments relate to methods and apparatusfor detection and separation of bacteria from a sample that remainviable. FIG. 29A shows that live vs. dead E. coli bacteria responddifferently to an electric field indicating that live and dead bacteriain a sample may be separated by tuning the frequency of the electricfield to a frequency of maximal capture. For instance, dead bacteriashow a peak response for capture around 10 MHz with essentially nocapture above 25 MHz, whereas live bacteria show a peak response forcapture around 40 MHz. Accordingly, to capture only live bacteria, theapplied electric field may be tuned to a frequency above 25 MHz, whichwould result in capture of the live bacteria, while dead bacteria wouldnot be captured. FIG. 29B shows results of testing multiple replicatesof live bacteria showing a consistent peak response of capture around 25MHz.

In a viability experiment, it was demonstrated that B. cereus suspendedin a tested buffer (PBS 1:1000 in deionized water) without staining withSybrGreen I is efficiently captured on a microfluidic chip designed inaccordance with the techniques describe herein when the electric fieldwas on. The tested buffer flowing through the chip was collected andplated on selected agar pates to calculate capture efficiency of thesystem. Bacteria captured on the electrodes were flushed with the testedbuffer solution while the electric field was still on. The electricfield was then turned off and the bacteria released from the electrodeswere collected and plated on agar plates to calculate release efficiencyand to confirm viability. The results of the experiment are shown inFIG. 30, which demonstrate that more that 60% of the bacteria capturedon the microfluidic chip and then release remained viable after release.

FIG. 31 shows a block diagram of an example computer system 3100 thatmay be used to implement embodiments of the technology described herein.The computing system 3100 may include one or more computer hardwareprocessors 3102 and non-transitory computer-readable storage media(e.g., memory 3104 and one or more non-volatile storage devices 3106).The processor(s) 3102 may control writing data to and reading data from(1) the memory 3104; and (2) the non-volatile storage device(s) 3106. Toperform any of the functionality described herein, the processor(s) 3102may execute one or more processor-executable instructions stored in oneor more non-transitory computer-readable storage media (e.g., the memory3104), which may serve as non-transitory computer-readable storage mediastoring processor-executable instructions for execution by theprocessor(s) 3102.

Having thus described several aspects and embodiments of the technologyset forth in the disclosure, it is to be appreciated that variousalterations, modifications, and improvements will readily occur to thoseskilled in the art. For example, while aspects of the present technologyrelate to an apparatus and methods for detection, separation,purification, and/or quantification of bacteria as described herein, theinventors have recognized that such apparatus and methods are broadlyapplicable to other organisms of interest, e.g. viruses, yeast, andaspects of the technology are not limited in this respect.

Such alterations, modifications, and improvements are intended to bewithin the spirit and scope of the technology described herein. Forexample, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the embodiments described herein. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments described herein. It is, therefore, to be understood thatthe foregoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto,inventive embodiments may be practiced otherwise than as specificallydescribed. In addition, any combination of two or more features,systems, articles, materials, kits, and/or methods described herein, ifsuch features, systems, articles, materials, kits, and/or methods arenot mutually inconsistent, is included within the scope of the presentdisclosure.

The above-described embodiments can be implemented in any of numerousways. One or more aspects and embodiments of the present disclosureinvolving the performance of processes or methods may utilize programinstructions executable by a device (e.g., a computer, a processor, orother device) to perform, or control performance of, the processes ormethods. In this respect, various inventive concepts may be embodied asa computer readable storage medium (or multiple computer readablestorage media) (e.g., a computer memory, one or more floppy discs,compact discs, optical discs, magnetic tapes, flash memories, circuitconfigurations in Field Programmable Gate Arrays or other semiconductordevices, or other tangible computer storage medium) encoded with one ormore programs that, when executed on one or more computers or otherprocessors, perform methods that implement one or more of the variousembodiments described above. The computer readable medium or media canbe transportable, such that the program or programs stored thereon canbe loaded onto one or more different computers or other processors toimplement various ones of the aspects described above. In someembodiments, computer readable media may be non-transitory media.

The above-described embodiments of the present technology can beimplemented in any of numerous ways. For example, the embodiments may beimplemented using hardware, software or a combination thereof. Whenimplemented in software, the software code can be executed on anysuitable processor or collection of processors, whether provided in asingle computer or distributed among multiple computers. It should beappreciated. that any component or collection of components that performthe functions described above can be generically considered as acontroller that controls the above-described function. A controller canbe implemented in numerous ways, such as with dedicated hardware, orwith general purpose hardware (e.g., one or more processor) that isprogrammed using microcode or software to perform the functions recitedabove, and may be implemented in a combination of ways when thecontroller corresponds to multiple components of a system.

Further, it should be appreciated that a computer may be embodied in anyof a number of forms, such as a rack-mounted computer, a desktopcomputer, a laptop computer, or a tablet computer, as non-limitingexamples. Additionally, a computer may be embedded in a device notgenerally regarded as a computer but with suitable processingcapabilities, including a Personal Digital Assistant (PDA), a smartphoneor any other suitable portable or fixed electronic device.

Also, a computer may have one or more input and output devices. Thesedevices can be used, among other things, to present a user interface.Examples of output devices that can be used to provide a user interfaceinclude printers or display screens for visual presentation of outputand speakers or other sound generating devices for audible presentationof output. Examples of input devices that can be used for a userinterface include keyboards, and pointing devices, such as mice, touchpads, and digitizing tablets. As another example, a computer may receiveinput information through speech recognition or in other audibleformats.

Such computers may be interconnected by one or more networks in anysuitable form, including a local area network or a wide area network,such as an enterprise network, and intelligent network (IN) or theInternet. Such networks may be based on any suitable technology and mayoperate according to any suitable protocol and may include wirelessnetworks, wired networks or fiber optic networks.

Also, as described, some aspects may be embodied as one or more methods.The acts performed as part of the method may be ordered in any suitableway. Accordingly, embodiments may be constructed in which acts areperformed in an order different than illustrated, which may includeperforming some acts simultaneously, even though shown as sequentialacts in illustrative embodiments.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The terms “substantially”, “approximately”, and “about” may be used tomean within ±20% of a target value in some embodiments, within ±10% of atarget value in some embodiments, within ±5% of a target value in someembodiments, within ±2% of a target value in some embodiments. The terms“approximately” and “about” may include the target value.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The invention claimed is:
 1. A bacterial separation system configured toseparate a first bacterial species and second bacterial species in asample including at least the first bacterial species, the secondbacterial species and other components, the system comprising: amicrofluidic chip including: a microfluidic passage comprising an inletand an outlet, wherein the microfluidic passage is configured to receivethe sample at the inlet of the microfluidic passage; a first electrodesystem disposed adjacent to a first portion of the microfluidic passageand between the inlet and the outlet of the microfluidic passage; and asecond electrode system disposed adjacent to a second portion of themicrofluidic passage and between the inlet and the outlet of themicrofluidic passage; at least one pump configured to pump the samplefrom the inlet of the microfluidic passage, past the first electrodesystem and the second electrode system, and to the outlet of themicrofluidic passage; and at least one signal generator electricallyconnected to the first electrode system and the second electrode system,wherein the at least one signal generator is configured to control thefirst electrode system and the second electrode system at least in partby: generating a first AC voltage to drive the first electrode system toproduce a first electric field having first characteristics that acts onthe sample when the at least one pump pumps the sample past the firstelectrode system within the first portion of the microfluidic passage;and generating a second AC voltage to drive the second electrode systemto produce a second electric field having second characteristics thatacts on the sample when the at least one pump pumps the sample past thesecond electrode system within the second portion of the microfluidicpassage, wherein the first characteristics are different than the secondcharacteristics, wherein the first characteristics are selected suchthat the first electric field exerts a positive dielectrophoresis forceon the first bacterial species sufficient to capture the first bacterialspecies on at least one first electrode in the first electrode systemwithout capturing the second bacterial species or the other componentsin the sample, and wherein the second characteristics are selected suchthat the second electric field exerts a positive dielectrophoresis forceon the second bacterial species sufficient to capture the secondbacterial species on at least one second electrode in the secondelectrode system without capturing the other components in the sample.2. The bacterial separation system of claim 1, wherein the at least onepump is coupled to the inlet of the microfluidic passage.
 3. Thebacterial separation system of claim 1, wherein the at least one pump iscoupled to the outlet of the microfluidic passage.
 4. The bacterialseparation system of claim 1, wherein a frequency of the second ACvoltage is higher than a frequency of the first AC voltage.
 5. Thebacterial separation system of claim 1, wherein the other componentsinclude a third bacterial species; the microfluidic chip furtherincludes a third electrode system disposed adjacent to a third portionof the microfluidic passage and between the inlet and the outlet of themicrofluidic passage; the at least one signal generator is furtherconfigured to control the third electrode system at least in part bygenerating a third AC voltage to drive the third electrode system toproduce a third electric field having third characteristics that acts onthe sample when the at least one pump pumps the sample past the thirdelectrode system within the third portion of the microfluidic passage;and the third characteristics are selected such that the third electricfield exerts a positive dielectrophoresis force on the third bacterialspecies sufficient to capture the third bacterial species on at leastone third electrode in the third electrode system without capturingcomponents of the other components other than the third bacterialspecies.
 6. The bacterial separation system of claim 5, wherein afrequency of the second AC voltage is higher than a frequency of thefirst AC voltage.
 7. The bacterial separation system of claim 6, whereina frequency of the third AC voltage is higher than the frequency of thesecond AC voltage.
 8. The bacterial separation system of claim 1,further comprising at least one optical system configured to capture oneor more first images of the at least one first electrode during captureof the first bacterial species and one or more second images of the atleast one second electrode during capture of the second bacterialspecies.
 9. The bacterial separation system of claim 8, furthercomprising: at least one computer configured to process the one or morefirst images to quantify an amount of bacteria of the first bacterialspecies captured by the at least one first electrode and/or process theone or more second images to quantify an amount of bacteria of thesecond bacterial species captured by the at least one second electrode.10. The bacterial separation system of claim 1, wherein the at least onefirst electrode comprises an array of first electrodes arranged in atleast one dimension along the microfluidic passage, and the at least onesecond electrode comprises an array of second electrodes arranged in theat least one dimension along the microfluidic passage.
 11. The bacterialseparation system of claim 10, wherein each of the array of firstelectrodes and the array of second electrodes is arranged in at leasttwo dimensions along the microfluidic passage.
 12. The bacterialseparation system of claim 1, wherein the at least one signal generatoris further configured to: generate, following capture of the firstbacterial species, a third AC voltage to drive the first electrodesystem to produce a third electric field having third characteristicswithin the first portion of the microfluidic passage, wherein the thirdcharacteristics are selected such that the third electric field exerts anegative dielectrophoresis force on the captured first bacterial speciessufficient to release the captured first bacterial species from the atleast one first electrode in the first electrode system.
 13. Thebacterial separation system of claim 12, wherein the at least one signalgenerator is further configured to: generate, following capture of thesecond bacterial species, a fourth AC voltage to drive the secondelectrode system to produce a fourth electric field having fourthcharacteristics within the second portion of the microfluidic passage,wherein the fourth characteristics are selected such that the fourthelectric field exerts a negative dielectrophoresis force on the capturedsecond bacterial species sufficient to release the captured secondbacterial species from the at least one second electrode in the secondelectrode system.
 14. The bacterial separation system of claim 1,wherein the at least one pump is disposed outside of a flow path of thesample.
 15. The bacterial separation system of claim 1, wherein the atleast one pump is disposed between the outlet of the microfluidicpassage and the first and second electrode systems.
 16. The bacterialseparation system of claim 1, wherein each of the first and secondelectrode systems comprise an electrode comprising a plurality ofconcentric arcs.
 17. The bacterial separation system of claim 16,wherein the plurality of concentric arcs of the respective electrodes ofthe first and second electrode systems are spaced equally from eachother.